Compositions and methods for treating muscular dystrophy and other disorders

ABSTRACT

The present invention provides compositions and methods of their use in treating muscular dystrophy and other disorders.

STATEMENT OF PRIORITY

This application is a continuation of U.S. patent application Ser. No.16/112,447, filed Aug. 24, 2018 (allowed), which is acontinuation-in-part application of U.S. patent application Ser. No.15/842,580, filed Dec. 14, 2017, now U.S. Pat. No. 10,245,235, issuedApr. 2, 2019, which claims the benefit, under 35 U.S.C. § 119(e), ofU.S. Provisional Application No. 62/435,442, filed Dec. 16, 2016 andU.S. Provisional Application No. 62/520,252, filed Jun. 15, 2017, theentire contents of each of which are incorporated by reference herein.

FIELD OF THE INVENTION

The present invention is directed to pharmaceutical formulations andmethods of use thereof in treating muscular dystrophy and otherdisorders.

BACKGROUND OF THE INVENTION

Dystroglycanopathies are a subset of muscular dystrophies characterizedby a secondary defect in glycosylation of alpha-dystroglycan (α-DG). Thediseases have been linked to autosomal-recessive mutations in at least18 different genes. They include fukutin-related protein (FKRP),fukutin, like-acetylglucosaminyltransferase (LARGE), POMGnT1, POMT1,POMT2, Isoprenoid Synthase Domain Containing (ISPD), Transmembraneprotein 5 (TMEM5), β1,3-N-acetylglucosaminyltransferasel (B3GNT1),glycosyltransferase-like domain containing 2 (GTDC2),β3-N-acetylgalactosaminyltransferase 2 (B3GALNT2), DOLK, GMPPB, DMP2,DMP3 and SGK196. Biochemical studies have established direct evidencefor involvement of a number of the genes in glycosylation modificationsof α-DG. Fukutin and Fukutin related protein (FKRP) genes have beenrecently proposed as Ribitol-5-P transferase that transfers thephosphorated ribitol to the core sugar chain of α-DG. LARGE protein actsas a bifunctional glycosyltransferase, xylosyltransferase andglucuronyltransferase, producing repeating units of[−3-xylose-α1,3-glucuronic acid-β1-] that is the functional glycan chainlinking cell membrane protein and extracellular matrix proteins. ThisLARGE glycan chain is linked to the core O-mannosyl glycans by tandemribitols. This linkage is critical for muscle health and lack of FKRPfunction as the result of gene mutations therefore prevents theproduction of functional glycosylation of α-DG, and disrupts normalinteraction between membrane and connective tissues, leading to musclefiber damage and muscular dystrophy.

Mutations in the FKRP gene cause a wide spectrum of disease from amilder form of limb-girdle muscular dystrophy (LGMD2I) to severeWalker-Warburg syndrome (WWS), muscle-eye-brain disease (MEB), andcongenital muscular dystrophy type 1D (MDC1D). However, little progresshas been made for the treatment of the diseases. There is no effectivetherapy available and only physical therapy and palliative care arebeing routinely provided as treatment.

The present invention overcomes previous shortcomings in the art byproviding pharmaceutical compositions and methods of their use intreating muscular dystrophy and other disorders.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a method of enhancingfunctional glycosylation of alpha-dystroglycan (α-DG) in a subjectwithout defects in dystroglycan-related genes and in need thereof,comprising administering to the subject an effective amount of ribitol,thereby restoring or enhancing functional glycosylation of α-DG in thesubject.

In a further aspect, the present invention provides a method of treatingmuscular dystrophy with the levels of ribitol and CDP-ribitol notaffected by the diseases, comprising administering to the subject aneffective amount of a ribitol, thereby treating the muscular dystrophyin the subject.

An additional aspect of this invention is a method of treating adisorder in a subject associated with a mutation in a fukutin relatedprotein (FKRP) gene, comprising administering to the subject aneffective amount of a ribitol, thereby treating the disorder in thesubject.

Further provided herein is a method of reducing and/or inhibiting theincidence of a neuronal migration abnormality or other disorder orsymptoms associated with a mutation in a FKRP gene in a subject known orsuspected to have a mutation in the FKRP gene, comprising administeringto the mother of the subject, during the subject's gestation in themother's uterus, an effective amount of a ribitol, thereby reducingand/or inhibiting the incidence of a neuronal migration abnormality, orother disorder or symptoms associated with a mutation in the FKRP geneof the subject.

In another aspect of this invention, a method is provided of treating orinhibiting the development of muscle weakness in a subject that is acarrier of a mutated FKRP gene, comprising administering to the subjectan effective amount of a ribitol, thereby treating muscle weakness,including but not limited to weakness of skeletal muscle, cardiac muscleand respiratory muscle, in the subject.

In another aspect of this invention, a method is provided of treating orinhibiting the development of muscle weakness in a subject that is notrelated to muscular dystrophy comprising administering to the subject aneffective amount of a ribitol, thereby treating muscle weakness,including but not limited to weakness of skeletal muscle, cardiac muscleand respiratory muscle, in the subject.

In addition, the present invention provides a method of treatingmuscular dystrophy that is not associated with a defect in glycosylationof α-DG in a subject, comprising administering to the subject aneffective amount of a ribulose, thereby treating the muscular dystrophythat is not associated with a defect in glycosylation of α-DG in thesubject.

Also provided herein is a method of treating a disorder associated witha mutation in a fukutin related protein (FKRP) gene in a subject,comprising administering to the subject an effective amount of aribulose, thereby treating the disorder associated with a mutation in afukutin related protein (FKRP) gene in the subject.

Furthermore, the present invention provides a method of reducing theincidence of a neuronal migration abnormality or other disorder orsymptoms associated with a mutation in a FKRP gene in a subject known orsuspected to have a mutation in the FKRP gene or without defect inglycosylation of α-DG, comprising administering to the mother of thesubject, during the subject's gestation in the mother's uterus, aneffective amount of a ribulose, thereby reducing the incidence of aneuronal migration abnormality, or other disorder or symptoms associatedwith a mutation in the FKRP gene of the subject.

In an additional aspect, the present invention provides a method oftreating or inhibiting the development of muscle weakness in a subjectthat is a carrier of a mutated FKRP gene or without defect inglycosylation of α-DG, comprising administering to the subject aneffective amount of a ribulose, thereby treating or inhibiting thedevelopment of muscle weakness.

The present invention is explained in greater detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Fluorescence-activated cell sorting (FACS) for the enhancedexpression of glycosylated alpha-DG after ribitol treatment. The breastcancer cell line MCF-7 was seeded in T25 culture flasks and cultured to75% confluence in DMEM 10% FBS, and then treated with 10 mM ribitol inthe same growth medium for 3 days. The cells were then collected bygentle scrapping and washed twice with PBS. The cells were resuspendedin 100 microliter PBS and stained with monoclonal antibody IIH6(Millipore EMD, 1:100 dilution) for 40 minutes and detected withsecondary Alexa 594-labeled goat anti-mouse IgM (Invitrogen). Thestained cells were washed and then FACS analyzed for the percentage ofpositive cells and the signal intensity (Alexa594.007). Untreated MCF-7cells cultured under the same conditions probed with secondary antibodyonly (Alexa594.005) and with both IIH6 and the secondary antibody(Alexa594.006) are used as controls.

FIGS. 2A-B. Induction of F-α-DG in cardiac and skeletal muscles of P448LMice treated with ribitol. Seven-week-old P448L mutant mice were givendrinking water only (control), or drinking water supplemented withribitol for 6 months. (2A) Western blot analysis and laminin overlayassay of protein lysates from heart, diaphragm (diaph), and tibialisanterior (TA) of control (−) or ribitol-treated (+) P448L, and C57 mice.F-α-DG was detected by blotting with IIH6C4 antibody, laminin overlayassay, and AF6868 antibody. Core of α-DG from cardiac tissue (heart) wasdetected by blotting with AF6868 antibody. Detection of GAPDH was usedas loading control. Arrow heads indicate laminin binding bands. Thestrong bands in laminin binding assay are endogenous laminin present inall samples. (2B) Quantification of IIH6C4 band intensity from westernblot. Values were normalized to GAPDH expression for each tissue andpresented as % expression compared to C57. Error bars representmean±SEM. Unpaired t test * p<0.05.

FIGS. 3A-B. Histopathology of muscle tissues from ribitol-treated P448Lmice. Seven-week-old P448L mutant mice were given drinking water only(control), or drinking water supplemented with ribitol for either 3months (3M) or 6 months (6M). (3A) Fiber size distribution of TA musclesof either control (n=3) or ribitol treated (n=4) P448L mutant mice, andwild-type C57 mice (n=4). (3B) Percentage of centrally-nucleated fibersin TA muscles of P448L mutant mice treated with ribitol for 3M and 6M,or aged matched control P448L mutant mice and wild-type C57 mice. Errorbars represent mean±SEM. Unpaired t test *p<0.05.

FIGS. 4A-B. Effect of ribitol treatment on histopathology of P448Lmutant mice. (4A) Fiber size distribution from quadriceps of eitherribitol treated (n=4) or age-matched control (n=3) P448L mutant mice,and wild-type C57 mice (n=4). (4B) Percentage of centrally-nucleatedfibers from quadriceps of 3M and 6M ribitol treated (n=4) or age-matchedcontrol (n=3) P448L mutant mice, and wild-type C57 mice (n=4). Errorbars represent mean±SEM. Unpaired t test *p<0.05.

FIG. 5. Effect of ribitol treatment on muscle fibrosis in P448L mutantmice. Seven-week-old P448L mutant mice were given drinking water only(control), or drinking water supplemented with ribitol for either 3months (3M) or 6 months (6M) Percentage of fibrotic areas quantifiedfrom Masson's Trichrome staining of heart, diaphragm and TA muscles ofeither ribitol-treated (for 3M and 6M) P448L mutant mice (n=4), orage-matched control P448L mutant mice (n=3) and wild-type C57 mice(n=4). Error bars represent mean±SEM. Unpaired t test *p<0.05.

FIGS. 6A-C. Evaluation of muscle and respiratory function onribitol-treated P448L mutant mouse. Seven-week-old P448L mutant micewere given drinking water only (control), or drinking water supplementedwith ribitol for either 3 months (3M) or 6 months (6M). (6A) Respiratoryfunction parameters from control or ribitol-treated P448L mice. (TV:tidal volume, EV: expiratory volume, MV: minute volume, PIF: peakinspiratory flow, PEF: peak expiratory flow, and f: breathingfrequency). (6B) Treadmill exhaustion test assessing the distance (m)and running time (min) until exhaustion covered by control orribitol-treated P448L mutant mice. (6C) Levels of FKRP transcript incardiac and skeletal muscles analyzed by quantitative real-time PCR.Error bars represent mean±SEM. Unpaired t test, * p<0.05. Significancein respiratory function noted with *. Significance in FKRP transcriptacross tissues noted with same letter.

FIG. 7. Effect of early 10% ribitol treatment on histopathology andmuscle function of P448L mutant mice. Mice were treated from pregnancyto 19 weeks of age. Control P448L mice were given drinking water only.(Panel A) H&E staining of tibialis anterior (TA) tissues from eithercontrol (P448L control), or ribitol-treated (P448L 10% ribitol) mutantmice. Percentage of centrally-nucleated fibers (% CNF) in TA musclestreated with 10% ribitol or aged matched control mutant mice and C57mice. Scale bar, 50 mm. (Panel B) Masson's Trichrome staining.Percentage of fibrotic areas quantified from the treated, age-matchedcontrol P448L mutant mice and C57 mice. (Panel C) Treadmill exhaustiontest shows significant improvement in distance (m) and running time(min) for the treated mice in comparison with control (Control).Unpaired t test *p<0.05. (Panel D) Grip strength test in control or 10%ribitol-treated mutant mice at the age of 18 weeks. Force (Unite) isnormalized to bodyweight (gr). (Panel E) Respiratory function fromcontrol and 10% ribitol-treated P448L mice at 18 weeks of age. (Ti:inspiratory time, MV: minute volume, EEP: expiratory pause, Penh:enhanced pause). Error bars represent mean±SEM. Unpaired t test *p<0.05.

FIG. 8. Detection of glycosylated α-DG in FKRPP448L mutant mouse (P448L)muscles treated with ribose in comparison with untreated FKRPP448L andnormal C57 mouse muscles. Glycosylated α-DG is detected with monoclonalantibody IIH6 specifically recognizing the functional sugar epitope ofthe α-DG. Positive signals present as membrane localized staining inalmost all muscle fibers of the muscle tissues from the ribose treatedmice whereas the same muscles from the untreated controls show only afew fibers with weak signal for glycosylated α-DG. Ribose treatment ofthe FKRPP448L mutant mice enhances expression of functionallyglycosylated α-dystroglycan. P448L control, mouse without treatment.P448L+ribose, mouse treated with 10 g/kg bodyweight ribose in drinkingwater for 1 month. C57, normal mouse. TA, Tibialis anterior muscle.Arrows indicate only a few fibers with the membrane staining forfunctionally glycosylated α-dystroglycan, whereas the majority of themuscle fibers are positive for the functionally glycosylatedα-dystroglycan in the ribose-treated muscles.

FIG. 9. Expression of glycosylated α-DG with IIH6 antibody in theepithelial cells after treatment with ribulose, CDP-ribitol and ribitol.The white lines represent membrane staining with the antibody. There areonly a few fibers in the control samples showing weak signalsrepresenting low levels of expression of functionally glycosylated α-DGwhereas positive signals are identified in the majority of the cellstreated with each of the three agents.

FIG. 10. Expression of glycosylated α-DG with IIH6 antibody in theepithelial cells after treatment with ribitol (lane 2) and ribulose(lane 3). Lane 1, control untreated cells; Lane 4, cells treated withribulose-5-phosphate; Lane 5, cells treated with glucose. Functionallyglycosylated alpha-DG is barely detectable in the control and the cellstreated with ribulose-5-phosphate and glucose.

FIG. 11. Ribulose treatment of the FKRPP448L mutant mice enhancesexpression of functionally glycosylated α-dystroglycan. P448L-control,mouse without treatment. P448L+Ribulose, mouse treated with 0.8 g/kgbodyweight ribulose intravenously and weekly for 1 month. C57, normalmouse. Signal of the membrane staining for functionally glycosylatedalpha-dystroglycan is hardly detectable whereas the majority of themuscle fibers are positive for the functionally glycosylatedalpha-dystroglycan in the ribulose-treated muscles.

FIG. 12. One month treatment with ribitol increases glycosylation ofα-DG in cardiac and skeletal muscles.

FIGS. 13A-B. Oral administration of ribitol increases levels ofribitol-5P and CDP-ribitol in muscle tissues. (13A) Increased levels ofmetabolites in heart and quadriceps of treated mice. (13B) Increasedlevels of CDP-ribitol in heart and quadriceps of treated mice.

FIGS. 14A-C. Long-term induction of functionally glycosylated α-DG byribitol in severely affected mutant mice. (14A) Treated mice showincrease in levels of F-α-DG after 6 months of treatments. (14B) Westernblot with IIH6C4 antibody. (14C) Treated mice show increase in levels ofribitol in cardiac muscle and diaphragm.

FIGS. 15A-C. 5% ribitol treatment alleviates dystrophic pathology inP448L mice and improves respiratory function. (15A) Hematoxylin andeosin (H&E) staining fibers in skeletal muscles of untreated mice. (15B)Decreased number of fibers with small diameter present in the quadricepsmuscles of treated animals. (15C) Percentage of CNF observed inribitol-treated and untreated mice.

FIGS. 16A-B. 5% ribitol treatment alleviates dystrophic pathology inP448L mice and improves respiratory function. (16A) Detection offibrotic tissue by Masson's Trichrome staining. (16B) Fibrosis of heartand diaphragm tissue in treated and untreated mice.

FIGS. 17A-C. Early treatment with 10% ribitol significantly improvesskeletal muscle function. (17A) Detection of F-α-DG in all skeletalmuscles and cardiac muscle of treated mice by immunohistochemistry.(17B) Western blot analysis to detect expression of F-α-DG. (17C)Expression of F-α-DG detected by western blots with antibody IIH6C4 inthe heart, diaphragm and limb muscle.

FIGS. 18A-E. Early treatment with 10% ribitol significantly improvesskeletal muscle function. (18A) Dystrophic pathology of treated mice.(18B) Reduction in fibrosis in cardiac muscle and diaphragm. (18C)Treadmill tests of the treated mice. (18D) Grip strength tests oftreated mice. (18E) Measurements in tidal volume (TV), minute volume(MV), end-expiratory and end-inspiratory pause (EEP and EIP,respectively) in treated and untreated mice.

FIG. 19. Model showing that the additional amount of ribitol allows themuscle fibers to produce higher than normal levels of FKRP substrate(CDP-ribitol), which enhances and partially compensates for the reducedfunction of mutant FKRP.

FIGS. 20A-B. Oral administration of ribitol increases levels ofribitol-5P and CDP-ribitol in muscle tissues. (20A) Development ofdetection method to detect metabolites. (20B) Standard curves for thequantification of metabolites.

FIGS. 21A-B. Oral administration of ribitol increases levels ofribitol-5P and CDP-ribitol in muscle tissues. (21A) LC/MS-MS method forthe detection of ¹³C-ribitol in cell samples. (21B) Detection ofendogenous analogs (ribitol, ribitol-5P and CDP-ribitol) in untreatedcells.

FIGS. 22A-B. Long-term induction of functionally glycosylated α-DG byribitol in mutant mice. (22A) Increase in levels of F-α-DG byimmunofluorescence with IIH6C4 after 3 months of treatment. (22B)Measured levels of mutant FKRP and LARGE transcripts by quantitativereal-time PCR in cardiac muscle, limb muscle and diaphragm.

FIGS. 23A-C. 5% ribitol treatment alleviates dystrophic pathology inmice and improves respiratory function. (23A) Hematoxylin and eosin(H&E) staining of skeletal muscles in untreated mice. (23B) Quantitativeanalysis from TA and quadriceps in mice after ribitol treatment. (23C)Percentage of CNF in ribitol-treated mice.

FIG. 24. Hematoxylin and eosin (H&E) staining of skeletal muscles intreated and the untreated P448L mice.

FIG. 25. Diaphragm of the untreated mice showing heavy fibrosis at the 3month time point and 6 months after study initiation.

FIGS. 26A-B. 5% ribitol treatment alleviates dystrophic pathology inP448L mice and improves respiratory function. (26A) Improvement in tidalvolume (TV), expiratory volume (EV) and minute volume (MV) in both 3 and6 month 5% ribitol-treated groups compared to the untreated P448L mice.(26B) Limb muscles function of treated mice.

FIG. 27. Pathology of heart and diaphragm of treated and untreated mice.

FIG. 28A. Early treatment with 10% ribitol significantly improvesskeletal muscle function. Treadmill tests showed improvement in expiredvolume (EV), relaxation time (RT) and enhanced pause (Penh).

FIG. 28B. Effects of ribitol treatment on body weight and histology ofliver, kidney and spleen. Body weight of treated and untreated male orfemale mice.

FIGS. 29A-B. Effects of ribitol treatment on body weight and histologyof liver, kidney and spleen. (29A) Histology of liver, kidney and spleenwith H&E staining. (29B) Biochemical analyses of serum markers for liverfunction and kidney function.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is explained in greater detail below. Thisdescription is not intended to be a detailed catalog of all thedifferent ways in which the invention may be implemented, or all thefeatures that may be added to the instant invention. For example,features illustrated with respect to one embodiment may be incorporatedinto other embodiments, and features illustrated with respect to aparticular embodiment may be deleted from that embodiment. In addition,numerous variations and additions to the various embodiments suggestedherein will be apparent to those skilled in the art in light of theinstant disclosure which do not depart from the instant invention.Hence, the following specification is intended to illustrate someparticular embodiments of the invention, and not to exhaustively specifyall permutations, combinations and variations thereof.

The disclosures of all patents, patent publications and non-patentdocuments cited herein are incorporated herein by reference in theirentirety.

The present invention is based on the unexpected discovery that ribitol,CDP-ribitol, ribose and/or ribulose can restore and/or enhancefunctional glycosylation of mainly alpha-dystroglycan (α-DG) in cellswithout defects in the genes related to muscular dystrophy and cellswith FKRP mutation. Yet, the same functional glycosylated epitope canalso modify other proteins. Therefore, restored or enhanced functionalglycosylation by ribitol and/or ribulose is not limited to α-DG and theuse of the phrase “functional glycosylation of α-DG” representsfunctional glycosylation of any protein after the use of ribitol and/orribulose.

Thus, in one embodiment, the present invention provides a method ofrestoring and/or enhancing functional glycosylation ofalpha-dystroglycan (α-DG) in a subject without defects indystroglycan-related genes and in need thereof, comprising administeringto the subject an effective amount of a ribitol, CDP-ribitol, riboseand/or ribulose, thereby restoring and/or enhancing functionalglycosylation of α-DG in the subject.

The present invention also provides a method of treating musculardystrophy without defects in dystroglycan-related genes (e.g., amuscular dystrophy that is not associated with a defect in glycosylationof α-DG) or defects or abnormalities in levels of the ribitol andCDP-ribitol in a subject, comprising administering to the subject aneffective amount of a ribitol, CDP-ribitol, ribose and/or ribulose,thereby treating the muscular dystrophy in the subject.

Furthermore the present invention provides a method of treating adisorder associated with (e.g., caused by or resulting from) a mutationin a fukutin related protein (FKRP) gene in a subject, comprisingadministering to the subject an effective amount of a ribitol,CDP-ribitol, ribose and/or ribulose, thereby treating the disorderassociated with a mutation in a fukutin related protein (FKRP) genedisorder associated with a mutation in a fukutin related protein (FKRP)gene in the subject.

In an additional embodiment, the present invention provides a method ofreducing the incidence of a neuronal migration abnormality or otherdisorder or symptoms associated with a mutation in a FKRP gene orwithout defect in a dystroglycan-related gene or in glycosylation ofα-DG, comprising administering to the mother of the subject, during thesubject's gestation in the mother's uterus, an effective amount of aribitol, CDP-ribitol, ribose and/or ribulose, thereby reducing theincidence of a neuronal migration abnormality, or other disorder orsymptoms associated with a mutation in the FKRP gene of the subject.

Additionally, the present invention provides a method of treating and/orinhibiting the development of muscle weakness in a subject that is acarrier of a mutated FKRP gene and/or without defect in adystroglycan-related gene and/or without defect in glycosylation ofα-DG, comprising administering to the subject an effective amount of aribitol, CDP-ribitol, ribose and/or ribulose, thereby treating muscleweakness. The muscle weakness can include but not limited to weakness ofskeletal muscle, cardiac muscle and/or respiratory muscle, in anycombination, in the subject.

The methods of this invention can also be used to treat non-musculardystrophy diseases for which restoration of and/or enhance glycosylationof α-DG would be beneficial and/or therapeutic. A nonlimiting example ofsuch a disease or disorder is a cancer that lacks, or expresses reducedlevels of glycosylated α-DG. The use of ribitol, CDP-ribitol, riboseand/or ribulose can restore and/or enhance levels of glycosylated α-DGor enhance glycosylation of other cell membrane proteins, thusinhibiting cancer cell growth and metastasis. Many cancer types,including breast cancer, prostate cancer, colon, head and neck cancersshow reduced expression of glycosylation of α-DG. Thus, in someembodiments, the present invention provides a method of treating cancerin a subject (e.g., a subject in need thereof), comprising administeringto the subject an effective amount of a ribitol, CDP-ribitol, riboseand/or ribulose, thereby treating the cancer in the subject. The presentinvention also provides a method of inhibiting and/or reducingmetastasis of cancer cells in a subject (e.g., a subject in needthereof), comprising administering to the subject an effective amount ofa ribitol, CDP-ribitol, ribose and/or ribulose, thereby inhibitingand/or reducing metastasis of the cancer cells in the subject.

Nonlimiting examples of a cancer that can be treated according to themethods of this invention include B cell lymphoma, T cell lymphoma,myeloma, leukemia, hematopoietic neoplasias, thymoma, lymphoma, sarcoma,lung cancer, liver cancer, non-Hodgkins lymphoma, Hodgkins lymphoma,uterine cancer, cervical cancer, endometrial cancer, adenocarcinoma,breast cancer, pancreatic cancer, colon cancer, anal cancer, lungcancer, renal cancer, bladder cancer, liver cancer, prostate cancer,ovarian cancer, primary or metastatic melanoma, squamous cell carcinoma,basal cell carcinoma, brain cancer, angiosarcoma, hemangiosarcoma, headand neck carcinoma, thyroid carcinoma, soft tissue sarcoma, bonesarcoma, testicular cancer, gastrointestinal cancer, and any othercancer now known or later identified (see, e.g., Rosenberg (1996) Ann.Rev. Med. 47:481-491, the entire contents of which are incorporated byreference herein).

In some embodiments of the methods of this invention, nonlimitingexamples of a disorder associated with a mutation in, or loss offunction of, the FKRP gene include limb-girdle muscular dystrophy(LGMD2I), Walker-Warburg syndrome (WWS), muscle-eye-brain disease (MEB),congenital muscular dystrophy type 1C (MDC1C), any other disorderassociated with a mutation in, or loss of function of, the FKRP gene,and any combination thereof.

In the methods of this invention, the ribitol can be, but is not limitedto, ribitol (adonitol) pentose alcohol, with or without modificationssuch as tri-acetylated ribitol (Ribitol(OAc)₃, per-acetylated ribitol(Ribitol(OAc)₅, a precursor thereof, such as ribose, a polysaccharidethereof, a phosphate form thereof, a non-phosphated form thereof, anyprecursor of a phosphate form, such as Ribose-5-P, any nucleotide formof ribitol (e.g., a nucleotide-alditol having cytosine or other bases asthe nucleobase with 1, 2 or 3 phosphate groups and ribitol as thealditol portion), such as CDP-ribitol, CDP-ribitol-OAc2 and anycombination or derivative or modification thereof.

A ribulose of this invention can be but is not limited to D-ribulose,D-erythro-pentulose, L-ribulose, L-erythro-pentulose, with and withoutmodification including but not limited to phosphorylation, and anycombination thereof.

The active compound of this invention (e.g., ribitol, CDP-ribitol,ribose and/or ribulose) can be present in a pharmaceutical formulationthat comprises substances and/or agents that are not natural products.As a nonlimiting example, the active compound (e.g., ribitol,CDP-ribitol, ribose and/or ribulose) of this invention can be present ina pharmaceutical composition with polyethylene glycol (PEG), which insome embodiments can have a molecular weight (MW) in a range of about200 to about 500. In some embodiments, a pharmaceutical composition ofthis invention can comprise glucose.

In some embodiments, the active compound of this invention (e.g.,ribitol, CDP-ribitol, ribose and/or ribulose) can comprise apolyalkylene glycol moiety coupled or linked thereto. “Polyalkyleneglycol” means straight or branched polyalkylene glycol polymersincluding, but not limited to, polyethylene glycol (PEG), polypropyleneglycol (PPG), and polybutylene glycol (PBG), as well as co-polymers ofPEG, PPG and PBG in any combination, and includes the monoalkylether ofthe polyalkylene glycol. Thus, in various embodiments of this invention,the polyalkylene glycol in the compositions of this invention can be,but is not limited to, polyethylene glycol, polypropylene glycol,polybutylene glycol, and any combination thereof.

In certain embodiments, the polyalkylene glycol of the composition ispolyethylene glycol or “PEG.” The term “PEG subunit” refers to a singlepolyethylene glycol unit, i.e., —(CH₂CH₂O)—. Thus, the active compoundcan be “pegylated.” In some embodiments, the PEG can have a molecularweight from about 10,000 g/mol to about 30,000 g/mol.

In some embodiments, the polyalkylene glycol (e.g., PEG) can benon-polydispersed, monodispersed, substantially monodispersed, purelymonodispersed, or substantially purely monodispersed.

“Monodispersed” is used to describe a mixture of compounds wherein about100 percent of the compounds in the mixture have the same molecularweight.

“Substantially monodispersed” is used to describe a mixture of compoundswherein at least about 95 percent of the compounds in the mixture havethe same molecular weight.

“Purely monodispersed” is used to describe a mixture of compoundswherein about 100 percent of the compounds in the mixture have the samemolecular weight and have the same molecular structure. Thus, a purelymonodispersed mixture is a monodispersed mixture, but a monodispersedmixture is not necessarily a purely monodispersed mixture.

“Substantially purely monodispersed” is used to describe a mixture ofcompounds wherein at least about 95 percent of the compounds in themixture have the same molecular weight and have the same molecularstructure. Thus, a substantially purely monodispersed mixture is asubstantially monodispersed mixture, but a substantially monodispersedmixture is not necessarily a substantially purely monodispersed mixture.

In further embodiments, the present invention provides a method ofenhancing expression of functional glycosylation of alpha-DG in asubject in need thereof, comprising administering to the subject aneffective amount of an active agent and/or composition of thisinvention. An example of a subject in need of such enhancement can be asubject that has muscle weakness without a defect in a gene known to beinvolved in glycosylsation.

The present invention further provides a method of treating a disorderassociated with a defect in glycosylation of alpha-DG, comprisingadministering to a subject that has or is suspected of having a disorderassociated with a defect in glycosylation of alpha-DG an effectiveamount of an active agent and/or composition of this invention. Asubject can be suspected of having a defect in glycosylation of alpha-DGif the subject has muscle weakness even in cases where genetic andbiochemical analyses of the subject have failed to identify a causativegene defect.

In additional embodiments, the present invention provides a method oftreating a disorder associated with muscle weakness, comprisingadministering to a subject that has or is suspected of having ofdeveloping a disorder associated with muscle weakness an effectiveamount of an active agent and/or composition of this invention. Muscleweakness can imply that a subject is not able to perform the dailyactivities that a normal person of similar gender, age and otherconditions would be expected to be capable of performing An example isthe loss of or lack of ability to climb stairs, run or hold an objectfor an extended period.

Further provided herein is a method of treating a disorder associatedwith a defect in glycosylation of alpha-DG caused by a mutation in theFKRP gene, comprising administering to a subject that has or issuspected of having a mutation in the FKRP gene an effective amount ofan active agent and/or composition of this invention. A mutation in anFKRP gene can be identified by genetic analysis of the nucleic acid of asubject.

In some embodiments of the methods of this invention, the ribitol,CDP-ribitol, ribose and/or ribulose can be administered or delivered toa subject in combination with (e.g., simultaneously, before and/orafter) CTP and/or any other nucleotide in an amount effective forenhancing the effect of the ribitol, CDP-ribitol, ribose and/or ribuloseon glycosylation of α-DG or other proteins. Furthermore, in the methodsof this invention, the ribitol, CDP-ribitol, ribose and/or ribulose canadministered with any other therapy (simultaneously, before and/orafter), such as steroid therapy and/or FKRP gene therapy to enhance orincrease the therapeutic effect.

Further aspects of this invention include the use of ribitol,CDP-ribitol, ribose and/or ribulose and/or a composition of thisinvention in the preparation of a medicament for carrying out themethods of this invention.

An additional aspect is the use of ribitol, CDP-ribitol, ribose and/orribulose and/or a composition of this invention for carrying out themethods of this invention.

The ribitol, CDP-ribitol, ribose and/or ribulose of this invention canbe in a composition comprising a pharmaceutically acceptable carrier.The therapeutically effective amount or dosage of the ribitol,CDP-ribitol, ribose and/or ribulose of this invention will varydepending on the subject's condition and therapeutic need, and will alsodepend, among other things, upon the effect or result to be achieved,the status of the subject and/or the route and/or mode of delivery. Insome embodiments, ribitol, CDP-ribitol, ribose and/or ribulose or anyother form(s) that can be converted to ribitol, or ribitol phosphate, ornucleotide-ribitol can be delivered orally in drinking water containingfrom about 0.1 to about 100% concentration of the drug as many times asdesirable, e.g., from about 1 time to about 100 times a day. The drugcan also be taken as pellet about 1 to about 10 times daily. The totalamount of the drug for daily use can be from about 0.001 g to about 500g depending on the nature and formulation of the ribitol, CDP-ribitol,ribose and/or ribulose, with enhanced effect, etc. The drug can be mixedor combined with any substance for improved delivery, absorption, etc.

Ribitols form in many plants and especially in the plant, Adonisvernalis, also known as spring pheasant's eye, or false hellebore, oryellow pheasant's eye and others. Adonis vernalis belongs to thebuttercup family Ranunculaceae. Plants containing ribitols can beadministered as the drug for treating FKRP-related diseases and subjectswith FKRP mutation and other diseases. Such plants can be directly usedas a food supplement, and/or ribitol can be extracted from the plantsfor administration as described herein.

Administration of the compound or composition of this invention may beby any suitable route, including but not limited to intrathecalinjection, subcutaneous, cutaneous, oral, intravenous, intraperitoneal,intramuscular injection, intra-arterial, intratumoral or any intratissueinjection, nasal, oral, sublingual, via inhalation, in an implant, in amatrix, in a gel, or any combination thereof.

Definitions.

As used herein, “a,” “an” or “the” can mean one or more than one. Forexample, “a” cell can mean a single cell or a multiplicity of cells.

Also as used herein, “and/or” refers to and encompasses any and allpossible combinations of one or more of the associated listed items, aswell as the lack of combinations when interpreted in the alternative(“or”).

The term “about,” as used herein when referring to a measurable valuesuch as an amount of dose (e.g., an amount of a fatty acid) and thelike, is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, ±0.5%,or even ±0.1% of the specified amount.

As used herein, the transitional phrase “consisting essentially of”means that the scope of a claim is to be interpreted to encompass thespecified materials or steps recited in the claim, “and those that donot materially affect the basic and novel characteristic(s)” of theclaimed invention. See, In re Herz, 537 F.2d 549, 551-52, 190 USPQ 461,463 (CCPA 1976) (emphasis in the original); see also MPEP § 2111.03.Thus, the term “consisting essentially of” when used in a claim of thisinvention is not intended to be interpreted to be equivalent to“comprising.”

“Subject” as used herein includes any animal in which functionalglycosylation of alpha-dystroglycan (α-DG) or other proteins isnecessary or desired. In some embodiments, the subject is any animalthat can receive a beneficial and/or therapeutic effect from restorationof functional glycosylation of alpha-dystroglycan (α-DG) and/orenhancement of glycosylation of α-DG. In some embodiments, the subjectis a mammal and in particular embodiments, the subject is a human of anyage, race, gender, or ethnicity, etc.

By the term “treat,” “treating” or “treatment of” (and grammaticalvariations thereof) it is meant that the severity of the subject'scondition is reduced, at least partially improved or ameliorated and/orthat some alleviation, mitigation or decrease in at least one clinicalsymptom is achieved and/or there is a delay or inhibition in theprogression of the disease or disorder.

“Treat,” “treating” or “treatment” as used herein also refers to anytype of action or administration that imparts a benefit to a subjectthat has a disease or disorder, including improvement in the conditionof the patient (e.g., reduction or amelioration of one or moresymptoms), healing, etc.

The terms “therapeutically effective amount,” “treatment effectiveamount” and “effective amount” as used herein are synonymous unlessotherwise indicated, and mean an amount of a compound, peptide orcomposition of the present invention that is sufficient to improve thecondition, disease, or disorder being treated and/or achieved thedesired benefit or goal (e.g., control of body weight). Those skilled inthe art will appreciate that the therapeutic effects need not becomplete or curative, as long as some benefit is provided to thesubject.

Determination of a therapeutically effective amount, as well as otherfactors related to effective administration of a compound of the presentinvention to a subject of this invention, including dosage forms, routesof administration, and frequency of dosing, may depend upon theparticulars of the condition that is encountered, including the subjectand condition being treated or addressed, the severity of the conditionin a particular subject, the particular compound being employed, theparticular route of administration being employed, the frequency ofdosing, and the particular formulation being employed. Determination ofa therapeutically effective treatment regimen for a subject of thisinvention is within the level of ordinary skill in the medical orveterinarian arts. In clinical use, an effective amount may be theamount that is recommended by the U.S. Food and Drug Administration, oran equivalent foreign agency. The amount of active ingredient that canbe combined with the carrier materials to produce a single dosage formvaries depending upon the subject being treated and the particular modeof administration.

As used herein, “modulate,” “modulates” or “modulation” refers toenhancement (e.g., an increase) or inhibition (e.g., diminished, reducedor suppressed) of the specified activity.

The term “enhancement,” “enhance,” “enhances,” or “enhancing” refers toan increase in the specified parameter (e.g., at least about a 1.1-fold,1.25-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 8-fold,10-fold, twelve-fold, or even fifteen-fold or more increase) and/or anincrease in the specified activity of at least about 5%, 10%, 25%, 35%,40%, 50%, 60%, 75%, 80%, 90%, 95%, 97%, 98%, 99% or 100%.

The term “inhibit,” “diminish,” “reduce” or “suppress” refers to adecrease in the specified parameter (e.g., at least about a 1.1-fold,1.25-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 8-fold,10-fold, twelve-fold, or even fifteen-fold or more increase) and/or adecrease or reduction in the specified activity of at least about 5%,10%, 25%, 35%, 40%, 50%, 60%, 75%, 80%, 90%, 95%, 97%, 98%, 99% or 100%.These terms are intended to be relative to a reference or control.

The above terms are relative to a reference or control. For example, ina method of enhancing glycosylation of α-DG in a subject of thisinvention by administering the ribitol, CDP-ribitol, ribose and/orribulose to the subject, the enhancement is relative to the amount ofglycosylation in a subject (e.g., a control subject) in the absence ofadministration of the ribitol, CDP-ribitol, ribose and/or ribulose.

“Isolated” as used herein means the ribitol of this invention issufficiently free of contaminants or cell components with which ribitolsmay occur. “Isolated” does not mean that the preparation is technicallypure (homogeneous), but it is sufficiently pure to provide the ribitolin a form in which it can be used therapeutically.

The term “prevent,” “preventing” or “prevention of” (and grammaticalvariations thereof) refers to prevention and/or delay of the onsetand/or progression of a disease, disorder and/or a clinical symptom(s)in a subject and/or a reduction in the severity of the onset and/orprogression of the disease, disorder and/or clinical symptom(s) relativeto what would occur in the absence of the methods of the invention. Theprevention can be complete, e.g., the total absence of the disease,disorder and/or clinical symptom(s). The prevention can also be partial,such that the occurrence of the disease, disorder and/or clinicalsymptom(s) in the subject and/or the severity of onset and/or theprogression is less than what would occur in the absence of the presentinvention.

A “prevention effective” amount as used herein is an amount that issufficient to prevent (as defined herein) the disease, disorder and/orclinical symptom in the subject. Those skilled in the art willappreciate that the level of prevention need not be complete, as long assome benefit is provided to the subject.

“Concurrently administering” or “concurrently administer” as used hereinmeans that the two or more compounds or compositions are administeredclosely enough in time to produce a combined effect (that is,concurrently may be simultaneously, or it may be two or more eventsoccurring within a short time period before and/or after each other,e.g., sequentially). Simultaneous concurrent administration may becarried out by mixing the compounds prior to administration, or byadministering the compounds at the same point in time but at differentanatomic sites and/or by using different routes of administration.

“Pharmaceutically acceptable” as used herein means that the compound orcomposition is suitable for administration to a subject to achieve thetreatments described herein, without unduly deleterious side effects inlight of the severity of the disease and necessity of the treatment.

Pharmaceutical Formulations.

The active compounds described herein may be formulated foradministration in a pharmaceutical carrier in accordance with knowntechniques. See, e.g., Remington, The Science and Practice of Pharmacy(21^(st) Ed. 2005). In the manufacture of a pharmaceutical formulationaccording to the invention, the active compound is typically admixedwith, inter alia, an acceptable carrier. The carrier must, of course, beacceptable in the sense of being compatible with any other ingredientsin the formulation and must not be deleterious to the subject. Thecarrier may be a solid or a liquid, or both, and is preferablyformulated with the compound as a unit-dose formulation, for example, atablet, which may contain from 0.01 or 0.5% to 95% or 99% by weight ofthe active compound. One or more active compounds may be incorporated inthe formulations of the invention, which may be prepared by any of thewell-known techniques of pharmacy comprising admixing the components,optionally including one or more accessory ingredients.

Furthermore, a “pharmaceutically acceptable” component such as a sugar,carrier, excipient or diluent of a composition according to the presentinvention is a component that (i) is compatible with the otheringredients of the composition in that it can be combined with thecompositions of the present invention without rendering the compositionunsuitable for its intended purpose, and (ii) is suitable for use withsubjects as provided herein without undue adverse side effects (such astoxicity, irritation, and allergic response). Side effects are “undue”when their risk outweighs the benefit provided by the composition.Non-limiting examples of pharmaceutically acceptable components includeany of the standard pharmaceutical carriers such as saline solutions,water, emulsions such as oil/water emulsion, microemulsions and varioustypes of wetting agents.

The formulations of the invention include those suitable for oral,rectal, topical, buccal (e.g., sub-lingual), vaginal, parenteral (e.g.,subcutaneous, intramuscular, intradermal, or intravenous), topical(i.e., both skin and mucosal surfaces, including airway surfaces) andtransdermal administration, although the most suitable route in anygiven case will depend on the nature and severity of the condition beingtreated and on the nature of the particular active compound which isbeing used.

Formulations suitable for oral administration may be presented indiscrete units, such as capsules, cachets, lozenges, or tablets, eachcontaining a predetermined amount of the active compound; as a powder orgranules; as a solution or a suspension in an aqueous or non-aqueousliquid; or as an oil-in-water or water-in-oil emulsion. Suchformulations may be prepared by any suitable method of pharmacy whichincludes the step of bringing into association the active compound and asuitable carrier (which may contain one or more accessory ingredients asnoted above). In general, the formulations of the invention are preparedby uniformly and intimately admixing the active compound with a liquidor finely divided solid carrier, or both, and then, if necessary,shaping the resulting mixture. For example, a tablet may be prepared bycompressing or molding a powder or granules containing the activecompound, optionally with one or more accessory ingredients.

Compressed tablets may be prepared by compressing, in a suitablemachine, the compound in a free-flowing form, such as a powder orgranules optionally mixed with a binder, lubricant, inert diluent,and/or surface active/dispersing agent(s). Molded tablets may be made bymolding, in a suitable machine, the powdered compound moistened with aninert liquid binder.

Formulations suitable for buccal (sub-lingual) administration includelozenges comprising the active compound in a flavoured base, usuallysucrose and acacia or tragacanth; and pastilles comprising the compoundin an inert base such as gelatin and glycerin or sucrose and acacia.

Formulations of the present invention suitable for parenteraladministration comprise sterile aqueous and non-aqueous injectionsolutions of the active compound(s), which preparations are preferablyisotonic with the blood of the intended recipient. These preparationsmay contain anti-oxidants, buffers, bacteriostats and solutes whichrender the formulation isotonic with the blood of the intendedrecipient. Aqueous and non-aqueous sterile suspensions may includesuspending agents and thickening agents. The formulations may bepresented in unit\dose or multi-dose containers, for example sealedampoules and vials, and may be stored in a freeze-dried (lyophilized)condition requiring only the addition of the sterile liquid carrier, forexample, saline or water-for-injection immediately prior to use.Extemporaneous injection solutions and suspensions may be prepared fromsterile powders, granules and tablets of the kind previously described.For example, in one aspect of the present invention, there is providedan injectable, stable, sterile composition comprising an activecompound(s), or a salt thereof, in a unit dosage form in a sealedcontainer. The compound or salt is provided in the form of alyophilizate which is capable of being reconstituted with a suitablepharmaceutically acceptable carrier to form a liquid compositionsuitable for injection thereof into a subject. The unit dosage formtypically comprises from about 10 mg to about 10 grams of the compoundor salt. When the compound or salt is substantially water-insoluble, asufficient amount of emulsifying agent which is physiologicallyacceptable may be employed in sufficient quantity to emulsify thecompound or salt in an aqueous carrier. One such useful emulsifyingagent is phosphatidyl choline.

Formulations suitable for rectal administration are preferably presentedas unit dose suppositories. These may be prepared by admixing the activecompound with one or more conventional solid carriers, for example,cocoa butter, and then shaping the resulting mixture.

Formulations suitable for topical application to the skin preferablytake the form of an ointment, cream, lotion, paste, gel, spray, aerosol,or oil. Carriers which may be used include petroleum jelly, lanoline,polyethylene glycols, alcohols, transdermal enhancers, and combinationsof two or more thereof.

Formulations suitable for transdermal administration may be presented asdiscrete patches adapted to remain in intimate contact with theepidermis of the recipient for a prolonged period of time. Formulationssuitable for transdermal administration may also be delivered byiontophoresis (see, for example, Pharmaceutical Research 3 (6):318(1986)) and typically take the form of an optionally buffered aqueoussolution of the active compound. Suitable formulations comprise citrateor bis\tris buffer (pH 6) or ethanol/water and contain from 0.1 to 0.2Mactive ingredient.

Further, the present invention provides liposomal formulations of thecompounds disclosed herein and salts thereof. The technology for formingliposomal suspensions is well known in the art. When the compound orsalt thereof is an aqueous-soluble salt, using conventional liposometechnology, the same may be incorporated into lipid vesicles. In such aninstance, due to the water solubility of the compound or salt, thecompound or salt will be substantially entrained within the hydrophiliccenter or core of the liposomes. The lipid layer employed may be of anyconventional composition and may either contain cholesterol or may becholesterol-free. When the compound or salt of interest iswater-insoluble, again employing conventional liposome formationtechnology, the salt may be substantially entrained within thehydrophobic lipid bilayer which forms the structure of the liposome. Ineither instance, the liposomes which are produced may be reduced insize, as through the use of standard sonication and homogenizationtechniques.

Of course, the liposomal formulations containing the compounds disclosedherein or salts thereof, may be lyophilized to produce a lyophilizatewhich may be reconstituted with a pharmaceutically acceptable carrier,such as water, to regenerate a liposomal suspension.

Other pharmaceutical compositions may be prepared from thewater-insoluble compounds disclosed herein, or salts thereof, such asaqueous base emulsions. In such an instance, the composition willcontain a sufficient amount of pharmaceutically acceptable emulsifyingagent to emulsify the desired amount of the compound or salt thereof.Particularly useful emulsifying agents include phosphatidyl cholines,and lecithin.

In addition to active compound(s), the pharmaceutical compositions maycontain other additives, such as pH-adjusting additives. In particular,useful pH-adjusting agents include acids, such as hydrochloric acid,bases or buffers, such as sodium lactate, sodium acetate, sodiumphosphate, sodium citrate, sodium borate, or sodium gluconate. Further,the compositions may contain microbial preservatives. Useful microbialpreservatives include methylparaben, propylparaben, and benzyl alcohol.The microbial preservative is typically employed when the formulation isplaced in a vial designed for multidose use. Of course, as indicated,the pharmaceutical compositions of the present invention may belyophilized using techniques well known in the art.

In some embodiments of this invention, the compound of this invention ispresent in an aqueous solution for subcutaneous administration. In someembodiments, the compound is provided as a lyophilized powder that isreconstituted and administered subcutaneously.

The present invention is illustrated in the following non-limitingexamples.

EXAMPLES Example 1

Currently, no disease specific treatment for FKRP-related diseases andany of glycosylation deficient muscular dystrophy is available.Glucocorticoid steroids (steroids) have been reported for thealleviation of disease symptoms with limited benefit, and largely basedon results from its reported uses in Duchenne muscular dystrophy.Therapeutic potential is believed to be achieved through itsanti-inflammatory effects. However, benefits of steroids to any musculardystrophy often last only a limited time period and are alwaysassociated with severe side effects including dramatic weight gain andreduction in bone mineral density, osteoporosis and growth retardation.Physical therapy and palliative care are routinely provided but onlyserve to relieve symptoms and are unable to delay disease progression.Currently there are several potential therapies including AAV genetherapy and gene correction in preclinical development fordystroglycanopathies, but none of them has enter the stage of clinictrials.

The use of viruses for gene and other expression vector delivery greatlyincrease their risks of immune response, non-target tissue expression,long-term toxicity of the overexpressed gene product and alteration ofgenomic sequence. The potential risks delay the progress in clinicaltrials. Further, their efficacy in clinic remains to be proved.

All available treatments, including use of steroids and physical therapycan only achieve relief of symptoms, such as inflammation and pain, butcannot effectively delay disease progression. Use of steroids is alwaysassociated with severe side effects, including dramatic weight gain andreduction in bone mineral density, causing osteoporosis and growthretardation. Both short term and long-term safety of the use ofextremely large quantity of virus for systemic delivery which isessential for achieving therapeutic value remain to be investigated andcannot be easily determined.

There is no similar approach of using sugar either as monosaccharides orpolysaccharides for treatment of muscular dystrophy. No sugar relatedtherapy for muscular dystrophy has ever been trialed to the inventor'sknowledge.

There is no similar approach of sugar either as monosaccharides orpolysaccharides for treatment of any cancer of which reducedglycosylation or loss of glycosylation of α-DG is related to tumorprogression and metastasis.

This invention identifies the use of a sugar, ribitol (adonitol) pentosealcohol, or any of its precursors, polysaccharides, phosphated ornon-phosphated forms, and nucleotide-forms (nucleotide-alditol havingcytosine or other bases as the nucleobase and ribitol as the alditolportion) for the treatment of subjects with muscular dystrophies and,especially for FKRP-mutation-related muscular dystrophies. Thistreatment achieves therapeutic effect likely through the restoration offunctional glycosylation of α-DG, which is lacking in subjects with FKRPmutations. This restoration reestablishes effective linkage between themuscle cells and the connective tissue surrounding fibers, thuspreventing damage to muscles. Thus, in some embodiments, this inventionapplies the sugar ribitol as the drug for treatment of musculardystrophy, including those types caused by mutations in the FKRP gene.Mutations in the FKRP gene cause muscular dystrophy with lack offunctional glycosylation of alpha-dystroglycan (α-DG) as thecharacteristic biochemical marker in the diseased tissues. Mouse modelswith the same FKRP mutations present the same biochemical feature aspatients' tissues, most prominently muscles (Chan et al.“Fukutin-related protein is essential for mouse muscle, brain and eyedevelopment and mutation recapitulates the wide clinical spectrums ofdystroglycanopathies” Hum Mol Genet 19(20):3995-4006 (2010); Blaeser etal. “Mouse models of fukutin-related protein mutations show a wide rangeof disease phenotypes” Hum Genet 132(8):923-934 (2013)). Usingimmunohistochemistry with a specific antibody, IIH6, to the functionalglycosylated α-DG, muscle fibers from both skeletal and cardiac musclesof FKRP P448L mutant mice produce a significantly decreased amount toalmost completely no functional glycosylated α-DG. This can also bedemonstrated by western blot detection. Histologically, the diseasedmuscles undergo continuous degeneration indicated by the presence ofdegenerating muscle fibers, variation in fiber size, presence ofcentrally nucleated fibers (a result of regeneration as a consequence ofmuscle damage) and inflammatory cells and increase in non-fiberconnective tissues. Surprisingly, feeding FKRP-P448L mutant dystrophicmice with ribitols either in drinking water or gavage, significantlyincreased the levels of functionally glycosylated α-DG in skeletalmuscles demonstrated by immunohistochemistry. More surprising, the levelof F-α-DG is even stronger in the cardiac muscle than in the skeletalmuscles, reaching signal intensity to levels similar to that detected innormal muscles. This is consistent in all mice treated with ribitols.

It is generally understood that IIH6 antibody detects functionalglycosylated α-DG, which representing the functional form of sugarmodification. However, the epitope detected by the IIH6 antibody may notbe limited to the α-DG. Further, other forms of functionallyglycosylated epitopes are likely present on α-DG and other proteins, andcan be enhanced by ribitol.

Ribitol treatment for 1.5 months improved muscle pathology with areduction in the number of centrally nucleated fibers (CNF) andinflammation. This can be most clearly demonstrated in the diseaseddiaphragm, which undergoes more severe and progressive degeneration andfibrosis.

The use of ribitol sugar as a drug provides the ideal means to treatthese diseases. No toxicity other than the effect of sugars is expected.This novel therapy not only can be universally applied to all patientswith FKRP mutations, it can also be applied to women who are pregnantwith a possible disease-carrying child. Carriers of an FKRP mutation,e.g., with a certain degree of muscle weakness, could benefit from theenhanced glycosylation of α-DG resulting from treatment with ribitol.

The drug can be administered to subjects with a single copy of FKRPmutation (heterozygotes, one copy of the FKRP gene is normal) andwithout obvious muscular dystrophy or other symptoms. The drug can bedelivered in any way described as for a muscular dystrophy patient,preferably with reduced dosage.

The drug can be administered to pregnant individuals with one fourthchance of having a baby that has FKRP-mutation related musculardystrophy. The drug can be delivered in any way described as for amuscular dystrophy patient.

Fluorescence-activated cell sorting (FACS) was used to show enhancedexpression of glycosylated alpha-DG after ribitol treatment. The breastcancer cell line MCF-7 was seeded in T25 culture flasks and cultured to75% confluence in DMEM 10% FBS, and then treated with 10 mM ribitol inthe same growth medium for 3 days. The cells were then collected bygentle scrapping and washed twice with PBS. The cells were resuspendedin 100 microliter PBS and stained with monoclonal antibody IIH6(Millipore EMD, 1:100 dilution) for 40 minutes and detected withsecondary Alexa 594-labeled goat anti-mouse IgM (Invitrogen). Thestained cells were washed and then FACS analyzed for the percentage ofpositive cells and the signal intensity (Alexa594.007). Untreated MCF-7cells cultured under the same conditions probed with secondary antibodyonly (Alexa594.005) and with both IIH6 and the secondary antibody(Alexa594.006) are used as controls. The cells treated with ribitol anddetected with the IIH6 antibody showed clearly higher levels ofglycosylated alpha-DG with 82% positive cells compared to the untreatedcontrols with only 45% positive cells (FIG. 1).

We first tried the overexpression of ISPD to FKRP P448L mutant mice tosee if increasing the activity of the gene can increase the levels ofCDP-ribitol and improve the glycosylation status of the α-DG in the FKRPmutant mouse models. However, no clear improvement has so far beenobserved. It is therefore a great surprise that supplementation withribitols can improve the glycosylation of α-DG significantly in animalswith both copies of the FKRP gene mutated. It is not understood howribitols can be delivered to diseased muscle effectively as no one hasever reported the use of ribitol for any medical treatment to theinventor's knowledge. It is even more surprising that the effect ofribitol on glycosylation of α-DG is greater in the cardiac muscle thanin the skeletal muscle.

Example 2

Mutations in Fukutin Related Protein (FKRP) gene causedystroglycanopathy characterized by defects in the O-mannosylation ofalpha dystroglycan (α-DG). FKRP functions as a ribitol-5-Phosphate(Rbo5P) transferase and is essential for the synthesis of functionallyglycosylated α-DG (F-α-DG). We tested the hypothesis that increase inlevels of ribitol, a precursor of FKRP substrate, could enhance thetransferase efficiency of mutant FKRPs, most of which retain at leastpartial function. We demonstrate that ribitol supplementationsystemically restored therapeutic levels of F-α-DG in both skeletal andcardiac muscles, and importantly improved muscle pathology and functionin an FKRP mutant model. Supplementation of ribitol or its derivativespresents a new approach to compensate for the defect in glycosylation ofα-DG with potential to treat more than 90% of FKRP dystroglycanopathies.

Mutations in the FKRP gene cause muscular dystrophies with a widevariation in severity from mild limb girdle muscular dystrophy (LGMD) 21to severe congenital muscular dystrophy (CMD), Walker-Warburg syndrome,and muscle-eye-brain disease. Mild LGMD2I is presented predominantly asmyopathy with progressive degeneration involving both skeletal andcardiac muscles. The continuous loss of muscle fibers and diminishingcapacity of regeneration eventually lead to the loss of muscle volumeand increase in fibrotic tissues. Consequently, patients gradually losemobility with impaired and ultimately failure of respiratory and cardiacfunctions. The severe forms of the disease can affect central nerve andoptical systems with developmental delay and mental retardation.Currently no treatment is available although several experimentaltherapies are being tested pre-clinically.

FKRP-related muscular dystrophies belong to a subset of the diseasecharacterized by a common secondary biochemical defect in theglycosylation of alpha-dystroglycan (α-DG). Alpha-DG is a peripheralmembrane protein extensively glycosylated with both N- and O-linkedglycans, the latter acting as a cellular receptor for laminin and otherextracellular matrix (ECM) proteins, including agrin, perlecan, neurexinand pikachurin. Importantly, the interaction of α-DG with ECM proteinsis critical for maintaining muscle integrity. The structure of thelaminin-binding O-mannosylated glycan on the functionally glycosylatedα-DG (F-α-DG) has recently been delineated with the following chain:(3G1cA-1-3Xyl-1)n-3G1cA-1-4Xyl-Rbo5P-1Rbo5P-3GalNAc-1-3G1cNAc-1-4(P-6)Man-1-Thr/ser.The entire process of the glycan chain extension pathway is completed bythe following proposed transferase activity, sequentially: POMT1 andPOMT2 catalyze the initial O-mannosylation of the proteins. Furtherextension of the sugar chain is carried out by POMGnT2 (GTDC2) andB3GALNT2, FKTN, FKRP, TMEM5 and B4GAT1 successively. Finally, LARGE actsas a bifunctional glycosyltrasferase having both xylosyltransferaase andglucuuronyltransferase activities, producing repeated units of3G1cA-1-3Xyl-1.

This study employed FKRP mutant mice containing a P448L mutation whichis associated with a severe dystrophic phenotype in clinic. Our resultsshow that ribitol supplementation can effectively restore therapeuticlevels of F-α-DG and significantly improve muscle pathology. Partialimprovement in muscle functions is also achieved without obvious sideeffects. This constitutes a potentially effective and safe treatment tothe diseases.

One month treatment with ribitol increases glycosylation of α-DG incardiac and skeletal muscles. In the pilot experiment, 4-week-old P448Lmice were treated with drinking water supplemented with 5% ribitol for 1month. Glycosylation of α-DG was analyzed by immunohistochemistry with amonoclonal antibody, IIH6C4, specifically recognizing thelaminin-binding epitopes of F-α-DG. As expected, F-α-DG was undetectablein skeletal muscles of the drinking water only control P448L mice,except for isolated or small clusters of revertant fibers in thediaphragm and tibialis anterior (TA) muscles. The revertant fibersexpressed variable levels of F-α-DG as reported previously. Occasionallyone or two fibers expressing F-α-DG over the entire cross section areawere also observed in cardiac muscle of the control P448L mice. Incontrast, oral ribitol supplementation visibly increased the signal ofF-α-DG in the heart, diaphragm and limb muscles of the treated mice.Membrane staining with IIH6C4 was observed in the majority of fibersfrom the limb muscles although most of them were only stained weakly.Areas of fibers with strong signals were also detected. The IIH6C4signals were consistently and clearly detected in the large proportionof diaphragm muscle fibers of the ribitol treated mice, although a smallproportion of fibers lacking detectable membrane staining remained.Interestingly, the signals for F-α-DG were easily distinguished and,particularly, more homogenous in the cardiac muscle than in the skeletalmuscles. However, the signals for F-α-DG in all the muscles ofribitol-treated mice were significantly weaker when compared to the samemuscle of wild-type C57 mice. We also identified that ribose treatmentin the same FKRP mouse model achieves enhancement of F-α-DG.

Prolonged treatment with ribitol maintains enhanced expression offunctionally glycosylated α-DG. To assess whether ribitol treatment canmaintain a long term effect on glycosylation of α-DG and whether sucheffect could improve disease pathology or slow progression, we extendedthe treatment of 5% ribitol in drinking water to the P448L mutant miceto 3 and 6 months and examined the expression of F-α-DG of the treatedmice in comparison with age-matched P448L controls. Consistent with the1 month treatment, all of the muscles from the 3 and 6 month treatedmice showed a clear increase in the levels of F-α-DG byimmunofluorescence staining with the IIH6C4. Tissue distribution of theenhanced F-α-DG remained similar to that in 1 month-treated muscles.Nearly all fibers in the cardiac muscle and the majority of fibers inboth diaphragm and limb muscles of treated mice were clearly positive.The signal intensity was homogenous in the cardiac muscle, but variedconsiderably in the skeletal muscles, especially in the diaphragm whereboth strong and weak expression were observed within vicinity. Themajority of muscle fibers in the TA muscles of treated mice containedweak signals and thus more homogeneous when compared to the signal inthe same muscle after 1 month ribitol treatment. Signal distribution andintensity for F-α-DG were generally similar in the same muscles between3 and 6 month ribitol-treated cohorts. Western blot analysis with theIIH6C4 antibody confirmed the enhanced expression of F-α-DG in themuscles of ribitol-treated P448L mice (FIG. 2A). Consistent with thesignal intensity shown by immunofluorescence detection after 6-monthtreatment, the levels of F-α-DG semi-quantified from the western blotwere distinctly detected in the cardiac muscle and diaphragm ofribitol-treated animals, reaching up to 6 and 8% of normal levels in C57mice, respectively (FIG. 2B). Only trace or indistinguishable signal wasdetected in the muscles of the control P448L mice. To further confirmthe ribitol-induced modification of α-DG, cardiac muscle samples werealso analyzed by western blot with the antibody AF6868, which detectsboth the functionally glycosylated form of α-DG (150-250 kDa as multiplebands) and the core α-DG representing species of non-functionallyglycosylated α-DG (core α-DG, 75-100 kDa). Ribitol-treated mice showeddetectable, although limited, increase of the higher molecular weightbands representing F-α-DG when compared to the P448L control (FIG. 2A).Similarly, laminin overlay assay demonstrated specific bands of theenhanced F-α-DG in ribitol-treated muscles, although only weakly,supporting the functionality of the ribitol-induced IIH6C4 positiveα-DG.

Ribitol treatment alleviates dystrophic pathology in FKRP P448L mutantmice. To evaluate whether the ribitol-induced increase in levels ofF-α-DG is sufficient to improve dystrophic pathology of muscles of theFKRP mutant mice, we performed histology examination of skeletal musclesafter 3 and 6 month treatments. H&E staining showed the presence oflarge areas of degenerating fibers, great variation in fiber sizes andcentrally nucleated fibers (CNF) in the skeletal muscles of the controlP448L mutant mice. This was also associated with focal inflammatoryinfiltrates. Treatment with 5% ribitol improved the dystrophic pathologyof TA and quadriceps muscles as evidenced by the diminished large fociof necrotic fibers and a more homogenously distributed fiber size.Quantitative analysis from TA and quadriceps showed a considerabledecrease in the number of fibers with small-diameters (newlyregenerated) indicating a decrease in degeneration after 3 and 6-monthribitol treatment when compared to control mice (FIG. 3A and FIG. 4A,for TA and quadriceps, respectively). However, no difference inpercentage of CNF was observed between ribitol treated and control P448Lmice (FIG. 3B and FIG. 4B, for TA and quadriceps, respectively).Notably, both 3 and 6 month ribitol treatments significantly decreasedareas of fibrotic tissue shown by Masson's Trichrome staining in the TAwhen compared to control mice. These results together suggest thatlimited enhancement in F-α-DG nevertheless improves pathology in thediseased limb muscles.

Importantly, ribitol treatment significantly improved pathology of thediaphragm. Large foci of degenerating fibers were common in the controldiaphragms but became rarely observed in all mice after 3 and 6 monthribitol treatments, although individual degenerating fibers remaineddetectable in some of the treated animals. Similarly, focal infiltrationwas also greatly diminished. The most striking improvement was thedegree of fibrosis. The diaphragm of the control mice showed clearlyvisible fibrosis at the 3 month time point (28.6%), reaching more than40% after 6 months from initiation of the study, as demonstrated byMasson Trichrome staining. However, the amount of fibrotic tissues inthe ribitol-treated cohort was significantly reduced to 11 and 18% after3 and 6 month treatment, respectively.

The cardiac muscle of the P448L mutant mice has limited pathology withonly a small increase in fibrotic area as disease progresses. H&Estaining did not show noticeable infiltration and degenerating fibers inboth the ribitol treated and the control mice. However, a significantreduction in fibrotic area was observed in the cardiac muscle of both 3and 6 month ribitol treated groups when compared to the controls (FIG.5).

Effect of ribitol treatment on respiratory and skeletal musclefunctions. To evaluate the effect of ribitol-induced improvement inF-α-DG on muscle functions, we conducted whole-body plethysmography forrespiratory function as well as treadmill exhaustion and grip strengthtests for skeletal muscle function at 3-month and 6-monthpost-initiation of ribitol treatment.

Plethysmography measured respiratory parameters including tidal volume(TV), expiratory volume (EV), minute volume (MV), peak inspiratory flow(PIF), peak expiratory flow (PEF), and breathing frequency (f) (FIG.6A). The results showed a trend of improvement in TV, EV and MV in both3 and 6 month ribitol-treated groups compared to the control P448Lmutant mice. Improvement was also observed in PIF and PEF at the 6 monthtime point. Furthermore, improvement in EV and MV reached statisticalsignificance in the 6 month ribitol-treated cohort. However, treadmilland grip force tests did not show a significant difference betweenribitol-treated and control P448L mice at both 3 and 6 month time points(FIG. 6B). These results are consistent with a more homogeneousenhancement in expression of F-α-DG and pronounced improvement inpathology in the diaphragm than in the limb skeletal muscles.

During the treatment period, we also weighed mice every 2 weeks untiltermination. There was no significant change in the percentage of weightgained between ribitol-treated and control P448L mutant mice althoughbody weight of the ribitol-treated female mice was slightly heavier.

Differential levels of FKRP expression in cardiac and skeletal muscles.The differential effect of ribitol treatment on expression of F-α-DG incardiac and skeletal muscles prompted us to assess whether variation inlevels of FKRP expression might be involved. Since no specific antibodyis currently available to reliably detect endogenous FKRP protein,quantitative real-time PCR using a mouse FKRP Taqman assay was used tomeasure the relative levels of FKRP mRNA in different muscles of bothwild type C57 and control P448L mutant mice. The heart muscle of bothmurine models demonstrated the highest expression in FKRP transcripts,significantly (10 times) higher than the skeletal muscles of the sameanimal model (FIG. 6C). Interestingly, FKRP transcript levels werehigher in the diaphragm than those detected in the quadriceps and TAmuscles in the P448L mutant mice. This tissue specific variation in FKRPexpression levels in both P448L mutant and wild-type models provides anexplanation for the differential effect of the ribitol treatment amongcardiac and skeletal muscles observed in the FKRP mutant mice.

Effect of early 10% ribitol treatment on histopathology and musclefunction of P448L mutant mice. Consistent with the enhancement on thebiochemical marker, dystrophic pathology in the 10% ribitol-treated micewas greatly alleviated with significantly fewer CNFs (FIG. 7A). Mostfibers of the limb muscles were highly homogenous in shape and size andonly a proportion of small CNFs were scattered within the diseasedmuscles. Notably, improvement in pathology with reduced infiltration andfiber size variation was also observed in the diaphragm. Furthermore,reduction in fibrosis was significant in cardiac muscle, and mostprominent in the diaphragm (FIG. 7B).

Importantly, early treatment with 10% ribitol significantly improvedskeletal muscle functions of the P448L mice. Treadmill tests showed thatboth running distance and time of the treated mice were significantlylonger than the age-matched control mice (FIG. 7C). Grip strength testsalso showed significant improvement on forelimb force from theribitol-treated mice compared to the control (FIG. 7D). Significantimprovement in respiratory functions was also demonstrated byplethysmography (FIG. 7E). Similar to the mutant mice treated for 6months with 5% ribitol, a trend of improvement in TV, EV and MV wasobserved, with MV reaching significant difference between the 10%ribitol treated and the control P448L mice. Furthermore, improvement onTi, EEP and Penh was also significant.

Despite significant advances in understanding the causes and clinicalmanifestation of the dystroglycanopathies, almost no progress has beenmade for the treatment of the diseases including those caused by FKRPmutations. Currently, physical therapy and other clinic managementroutinely provided to patients only serve as palliative care. The onlyoption of pharmacological intervention available is glucocorticoidsteroids which are being used anecdotally based on reported benefitsfrom other muscular dystrophies, especially Duchenne muscular dystrophy(DMD). However, clinic efficacy of steroids has not been systematicallyinvestigated for dystroglycanopathies and severe side effects includingimmune suppression and reduction in bone mineral density are reported indystroglycanopathy mouse model with FKRP mutations. Therefore, there isan urgent need for developing experimental therapies to the diseases.Here we show that ribitol, a natural sugar compound present in someplants and animals and considered as a metabolic intermediate orend-product, can effectively restore therapeutic levels of F-α-DG and,more importantly, ameliorate dystroglycanopathy caused by the FKRP P448Lmutation which is associated with severe CMD disease phenotype inclinic. Our results demonstrate a potentially safe and effective newtreatment for the majority of FKRP dystroglycanopathies and raise thepotential of developing similar approaches for other diseases associatedwith aberrant O-mannosylation of α-DG.

Animal care. All animal studies were approved by the InstitutionalAnimal Care and Use Committee (IACUC) of Carolinas Medical Center. Allmice were housed in the vivarium of Carolinas Medical Center accordingto animal care guidelines of the institute. Animals were ear taggedprior to group assignment. Food and water were available ad libitumduring all phase of the study. Body weight was measured every 2 weeks.

Mouse model and experimental procedure. FKRP P448L mutant mice weregenerated by the McColl-Lockwood Laboratory of Muscular DystrophyResearch. These mice contain a homozygous missense mutation (c.1343C>T,p.Pro448Leu) in the FKRP gene with the floxed neomycin resistant(Neo^(T)) cassette removed from the insertion site. C57BL/6(wild-type/C57) mice were purchased from Jackson Laboratory.

Ribitol was purchased from Sigma (A5502 Adonitol, ≥99%, Sigma, St.Louis) and dissolved in drinking water to the final concentration of 5%.P448L mutant mice aged at 4 weeks were treated with 5% ribitol drinkingwater for 1 month and P448L mice aged at 7 weeks were treated with 5%ribitol drinking water for 3-months and 6-months with 4-5 mice for eachcohort. The levels of drinking solution in the feeding bottles waschecked every day. Age-matched P448L mutant and wild-type C57BL/6 micewere used as controls. The animals were terminated at the end of eachtreatment time point and tissues including heart, diaphragm, TA,quadriceps, liver and kidney were collected for analyses.

Immunohistochemical and western blot analysis. Tissues were dissectedand snap-frozen in dry-ice-chilled-2-methylbutane. Forimmunohistochemical detection of functionally glycosylated α-DG, crosssections of 6 μm thickness were first fixed in ice cold Ethanol:Aceticacid (1:1) for 1 min, blocked with 10% normal goat serum (NGS) in1×Tris-buffer saline (TBS) for 30 min at room temperature, and incubatedovernight at 4° C. with primary mouse monoclonal antibody IIH6C4 (EMDMillipore) (1:500) against α-DG. Negative controls received 10% normalgoat serum in 1×TBS only. Sections were washed and incubated withsecondary AlexaFLuor 488 goat anti-mouse IgM (Invitrogen) (1:500) atroom temperature for 1 hr. Sections were washed and finally mounted withfluorescence mounting medium (Dako) containing 1×DAPI(4′,6′-diamidino-2-phenylindole) for nuclear staining Immunofluorescencewas visualized using an Olympus BX51/BX52 fluorescence microscope(Opelco) and images were captured using the Olympus DP70 digital camerasystem (Opelco).

For western blot analysis, tissues were homogenized in extraction buffer(50 mM Tris-HCl pH 8.0, 150 mM NaCl, and 1% Triton X-100), supplementedwith 1× protease inhibitor cocktail (Sigma-Aldrich). Proteinconcentration was quantified by Bradford assay (Bio-Rad DC proteinassay). Eighty μg of protein was loaded on an 8-16% Tris-glycinepolyacrylamide gel (Life Technologies) and immunoblotted. Nitrocellulosemembranes (Bio-Rad) were blocked with 3% milk in 1×PBS for 2 hr at roomtemperature and then incubated with the following primary antibodiesovernight at 4° C.: IIH6C4 (1:2000), AF6868 (R&D Systems) (1:1000),GAPDH (Thermo Fisher) (1:1000). Appropriate horseradish peroxidase(HRP)-conjugated secondary antibodies were incubated for 2 hr at roomtemperature. All blots were developed by electrochemiluminescenceimmunodetection (PerkinElmer). For IIH6C4 band quantification fromwestern blot the GelAnalyzer 2010a software was used. For lamininoverlay assay, nitrocellulose membranes were blocked with lamininoverlay buffer (10 mM ethanolamine, 140 mM NaCl, 1 mM MgCl2, and 1 mMCaCl₂, pH 7.4) containing 5% nonfat dry milk for 1 hr at 4° C. followedby incubation with laminin from Engelbreth-Holm-Swarm murine sarcomabasement membrane (L2020) (Sigma) at a concentration of 2 μg/mlovernight at 4° C. in laminin overlay buffer. Membranes were thenincubated with rabbit anti-laminin antibody (Sigma) (1:1500) followed bygoat anti-rabbit HRP-conjugated IgG secondary antibody (Santa CruzBiotechnology) (1:3000).

Quantitative reverse transcriptase PCR assay. Tissue was extracted fromshavings of snap frozen muscles of three mice for each group (C57 andP448L). RNA was extracted using TRIzol (Invitrogen) following thesupplied protocol. Final RNA pellet was re-suspended in 20 μlRNAse-nuclease free water. Final RNA concentration was determined usingNanodrop 2000c. One μg of RNA was subsequently converted to cDNA usingthe High-Capacity RNA-to-cDNA™ Kit (Applied Biosystems) following thesupplied protocol. cDNA was then used for quantitative real-time PCRusing the mouse FKRP-FAM Taqman assay (Mm00557870_m1) with primerlimited GAPDH-VIC (Mm99999915_g1) as the internal control and TAQMAN®Universal Master Mix II, with UNG (Life Technologies). Quantitative realtime PCR was run on the BioRad CFX96 TOUCH™ Real-Time PCR DetectionSystem (BioRad) following the standard real time PCR conditionssuggested for taqman assays. Results of FKRP transcript were calculatedand expressed as 2{circumflex over ( )}-ΔΔCt and compared across tissuesand animals.

Histopathological and morphometric analysis. Frozen tissues wereprocessed for hematoxylin and eosin (H&E) and Masson's Trichromestaining following standard procedures. Muscle cross-sectional fibers ofequivalent diameter were determined from tibialis anterior andquadriceps stained with H&E using MetaMorph v7.7 Software (MolecularDevices). Percentage of centrally nucleated myofibers was manuallyquantified from the same tissue sections stained with H&E. Fibrotic arearepresented by blue staining in the Masson's trichrome stained sectionswas quantified from heart, diaphragm, tibialis anterior and quadricepsusing ImageJ software. For all the morphometric analyses, a total of 300to 400 fibers from two representative 20× magnification images per eachmuscle per animal was used.

Muscle function tests. For treadmill exhaustion test, mice were placedon the belt of a five-lane-motorized treadmill (Columbus Instruments)supplied with shock grids mounted at the back of the treadmill, whichdelivered a 0.2-mA current to provide motivation for exercise.Initially, the mice were subjected to an acclimation period (time, 5min; speed, 8 cm/s, and 0° incline) Immediately after acclimationperiod, the test commenced with speed increases of 2 cm/s every minuteuntil exhaustion. The test was stopped and the time to exhaustion wasdetermined when the mouse remained on the shock grid for 5 s withoutattempting to re-engage the treadmill.

For grip force test, forelimb and hind limb in peak torque (g) wasmeasured by a grip strength meter (Columbus Instruments). For forelimbforce, the animal was held so that only the forelimb paws grasp thespecially designed mouse flat mesh assembly and the mouse pulled backuntil their grip is broken. The force transducer retains the peak forcereached when the animal's grip is broken and is recorded from a digitaldisplay. For hind limb force, an angled mesh assembly was used. Micewere allowed to rest on the angled mesh assembly, facing away from themeter with its hind limbs at least one-half of the way down the lengthof the mesh. The mouse tail was pulled directly toward the meter andparallel to the mesh assembly. During this procedure, the mice resist bygrasping the mesh with all four limbs. Pulling toward the meter wascontinued until the hind limbs released from the mesh assembly. Fivesuccessful hind limb and forelimb force measurements within 2 minuteswere recorded. The average value was used for analysis. Forelimb andhind limb force are presented as values of KGF (kilogram-force) unitsnormalized to bodyweights (gr) as “Units/gr”. All muscle function testswere performed 2 weeks before euthanasia.

Whole body plethysmography. Respiratory functional analysis inconscious, freely moving mice was measured using a whole bodyplethysmography technique. The plethysmograph apparatus (emkaTechnologies, Falls Church, Va.) was connected to a ventilation pump forthe purpose of maintaining a constant air flow, a differential pressuretransducer, a usbAMP signal amplifier, and a computer running EMKA iox2software with the respiratory flow analyzer module, which was used todetect pressure changes due to breathing and recording the transducersignal. An initial amount of 20 mL of air was injected and withdrawn viaa 20 mL syringe into the chamber for the purpose of calibration. Micewere placed inside the “free moving” plethysmograph chamber and allowedto acclimate for 5 min in order to minimize any effects of stressrelated changes in ventilation. Resting ventilation was measured for aduration of 15 min after the acclimation period. Body temperatures ofall mice were assumed to be 37° C. and to remain constant during theventilation protocol.

Statistical analysis. All data are expressed as mean±SEM unless statedotherwise. Statistical analyses were performed with GraphPad Prismversion 7.01 for Windows (GraphPad Software). Individual means werecompared using multiple t tests. Differences were considered to bestatistically significant at p≤0.05 (*).

Although the present invention has been described with reference tospecific details of certain embodiments thereof, it is not intended thatsuch details should be regarded as limitations upon the scope of theinvention except as and to the extent that they are included in theaccompanying claims.

Throughout this application, various patents and non-patent publicationsare referenced. The disclosures of these patents and publications intheir entireties are hereby incorporated by reference into thisapplication in order to more fully describe the state of the art towhich this invention pertains.

Example 3. Testing of Ribose to Restore Functional Glycosylation of α-DGin Mutant Mouse Model

Detection of glycosylated α-DG in FKRPP448L mutant mouse (P448L) musclestreated with ribose in comparison with untreated FKRPP448L and normalC57 mouse muscles. Glycosylated α-DG is detected with monoclonalantibody IIH6, which specifically recognizes the functional sugarepitope of the α-DG. Positive signals are present as membrane localizedstaining in almost all muscle fibers of the muscle tissues from theribose treated mice whereas the same muscles from the untreated controlsshow only a few fibers with weak signal for glycosylated α-DG (FIG. 8).

Cross sections were first fixed in ice cold Ethanol:Acetic acid (1:1)for 1 min, blocked with 10% normal goat serum (NGS) in 1×Tris-buffersaline (TBS) for 30 min at room temperature, and incubated overnight at4° C. with primary antibody IIH6C4 (EMD Millipore) (1:500) against α-DG.Negative controls received 10% normal goat serum in 1×TBS only. Sectionswere washed and incubated with secondary AlexaFLuor 488 goat anti-mouseIgM (Invitrogen) (1:500) at room temperature for 2 hr. Sections werewashed and finally mounted with fluorescence mounting medium (Dako)containing 1×DAPI (4′,6′-diamidino-2-phenylindole) for nuclear staining.Immunofluorescence was visualized using an Olympus BX51/BX52fluorescence microscope (Opelco) and images were captured using theOlympus DP70 digital camera system (Opelco). Slides were examined in ablinded manner by the investigator.

Example 4. Treatment of Epithelial Cells with Ribulose, CDP-Ribitol, andRibitol

Expression of glycosylated α-DG with IIH6 antibody in the epithelialcells after the treatment with ribulose, CDP-ribitol and ribitol wereobserved using spectroscopic methods. The white lines represent themembrane staining with the antibody. There are only a few fibers in thecontrol samples showing weak signals representing low levels ofexpression of functionally glycosylated α-DG whereas positive signalsare identified in the majority of the cells treated with each of all thethree agents (FIG. 9). Cells are grown in culture medium and activereagents were added one day later and culture for further 3 days withthe described concentration. The cells were then washed, fixed andstained with IIH6 antibody for the expression of glycosylated α-DG. Thepositive signals present as membrane staining (white lines and circleshere) which are only detected in the majority of the treated samples,but hardly seen in the control.

Example 5. Treatment of Epithelial Cells with Ribulose and Ribitol

Expression of glycosylated α-DG with IIH6 antibody in the epithelialcells after the treatment with ribitol (lane 2) and ribulose (lane 3)were observed visually. Lane 1, control untreated cells; Lane 4, cellstreated with ribulose-5-phosphate; Lane 5, cells treated with glucose.Functionally glycosylated alpha-DG is barely detectable in the controland the cells treated with ribulose-5-phosphate and glucose (FIG. 10).Cells are grown in culture medium and active reagents were added one daylater and culture for further 3 days with the active agent. The cellswere then collected, homogenized, blotted onto membrane and detectedwith IIH6 antibody for the expression of glycosylated α-DG. The blacklarge spot represents the expression of the glycosylated α-DG, withhigher intensity indicating higher levels.

Example 6. Ribulose Treatment of FKRP448L Mutant Mice

Ribulose treatment of the FKRPP448L mutant mice enhances expression offunctionally glycosylated α-dystroglycan. P448L-control, mouse withouttreatment. P448L+Ribulose, mouse treated with 0.8 g/kg bodyweightribulose intravenously and weekly for 1 month. C57, normal mouse. Signalof the membrane staining for functionally glycosylated α-dystroglycan ishardly detectable whereas the majority of the muscle fibers are positivefor the functionally glycosylated α-dystroglycan in the ribulose-treatedmuscles (FIG. 11). Animals were treated with ribulose for 4 weeks withweekly injection of 0.8 g/kg bodyweight. Animal tissues were collectedat the end of 4 weeks and processed for sections, fixed, stained withthe IIH6 antibody and visualized as membrane staining for theglycosylated α-DG. Untreated age-matched P448L untreated and wild-typeC57BL/6 mice were used as controls. There is wide spread staining in themajority of muscle fibers in the ribulose treated samples whereas barelyany signal is observed in the control samples. The signals in the C57mice are the strongest.

Example 7. Ribitol Restores Functionally Glycosylated α-Dystroglycan andImproves Muscle Functions in FKRP Dystroglycanopathy

In this study, we tested our hypothesis in the FKRP mutant micecontaining P448L mutation which is associated with CMD in clinic. Ourresults show that ribitol treatment increases levels of ribitol-5P andCDP-ribitol in muscle tissue and can effectively restore therapeuticlevels of F-α-DG both before and after the onset of the diseasephenotype. This results in significant improvement in muscle pathologyand functions. Moreover, no side effects were detected in histology andfunctions of liver and kidney, muscle development, body weight andbehavior of the animals. To the best of our knowledge, this is the firstdemonstration that a pentose alcohol ribitol constitutes a potentiallyeffective and safe treatment to FKRP dystroglycanopathies.

One month treatment with ribitol increases glycosylation of α-DG incardiac and skeletal muscles. We have previously reported a FKRP mousemodel containing a P448L mutation (P448L) with onset of the dystrophicpathology as early as 3 weeks of age. In the pilot experiment,4-week-old P448L mice were treated with drinking water supplemented with5% ribitol for 1 month. Glycosylation of α-DG was analyzed byimmunohistochemistry with a monoclonal antibody, IIH6C4, specificallyrecognizing the laminin-binding epitopes of F-α-DG. Consistent withearly reports, F-α-DG was undetectable in cardiac and skeletal musclesof the untreated P448L mice given drinking water only, except forisolated small clusters of revertant fibers in skeletal muscles, and oneor two fibers expressing F-α-DG in cardiac muscle. (FIG. 12). Incontrast, oral 5% ribitol treatment visibly increased F-α-DG in theheart, diaphragm and limb muscles. The signals of F-α-DG wereconsistently and clearly detected in the large proportion of diaphragmmuscle fibers of the ribitol-treated mice. Interestingly, the signalsfor F-α-DG were easily detected with higher homogeneity in the cardiacmuscle than in the skeletal muscles. Signals for F-α-DG in all themuscles of ribitol-treated mice were in general weaker when compared tothe same muscle of C57 mice.

Oral administration of ribitol increases levels of ribitol-5P andCDP-ribitol in muscle tissues. To evaluate whether oral administrationof ribitol increases levels of ribitol-5P and CDP-ribitol in cardiac andskeletal muscles of mutant mice, we analyzed and quantified ribitol,ribitol-5P and CDP-ribitol in muscle tissues by LC/MS-MS. Ribitol(Sigma) as well as synthesized ribitol-5P and CDP-ribitol (Z-Biotech)were used to develop the detection method and to establish the standardcurves for the quantification of the metabolites (FIGS. 20a and 20b ,respectively). Endogenous levels of ribitol, ribitol-5P and CDP-ribitolwere similar between untreated mutant P448L and C57 control mice (FIG.13b ). The three metabolites showed increased levels in heart andquadricep of the 5% ribitol-treated mice compared to untreated P448Lmice (FIG. 13a and FIG. 13b ). Levels of CDP-ribitol were at least4-fold higher in heart and quadriceps of treated mice when compared tountreated and the difference of ribitol-5P and CDP-ribitol levels werestatistically significant in both heart and quadricep (FIG. 13b ). Thelevels of the metabolites were apparently higher in the heart tissuesthan in the skeletal muscles.

To address the question whether the orally administrated ribitol is, infact, converted to ribitol-5P and CDP-ribitol, we treated differentiatedC2C12 myotubes with isotopically labeled ¹³C₅-ribitol in vitro.¹³C₅-ribitol (Omicron Biochemicals, Inc.) was used to develop theLC/MS-MS method for detection of ¹³C-ribitol in cell samples (FIG. 21a). The MRM (multi-reaction monitoring) methods for ¹³C-ribitol-5P andCDP-¹³C-ribitol were inferred from their non-labeled analogs (mass+5amu). The LC/MS-MS analysis from the untreated cells showed low levelsof endogenous ribitol, ribitol-5P and CDP-ribitol and absence of¹³C-labeled analogs. However, the cells treated with ¹³C-ribitol showedclearly elevated levels of ¹³C-ribitol-5P and CDP-¹³C-ribitol as well as¹³C-ribitol, but only background levels of endogenous analogs (ribitol,ribitol-5P and CDP-ribitol) as detected in the untreated cells (FIG. 21b). All together, these results confirm that exogenous ribitol can beconverted to ribitol-5P and most importantly CDP-ribitol, the FKRPsubstrate for F-α-DG synthesis.

Long-term induction of functionally glycosylated α-DG by ribitol inseverely affected mutant mice. To assess whether ribitol treatment canmaintain a long-term effect on glycosylation of α-DG in mutant micealready exhibiting severe dystrophic phenotype, we treated the P448Lmice at the age of 7 weeks with 5% ribitol in drinking water for up to 3and 6 months. Consistent with the 1 month treatment, all muscles fromboth cohorts of treated mice showed a clear increase in the levels ofF-α-DG by immunofluorescence with IIH6C4 (FIG. 14a and FIG. 22a for 6months and 3 months-treatments, respectively). Nearly all fibers in thecardiac muscle, and a majority of fibers in both diaphragm and limbmuscles, were positive for F-α-DG (FIG. 14a ). Signal distribution andintensity for F-α-DG were generally similar in the same muscles between3 and 6 month ribitol-treated cohorts. The enhanced expression of F-α-DGby ribitol was further confirmed by western blot analysis with IIH6C4antibody, reaching up to 14 and 17% of normal levels in the cardiacmuscle and diaphragm, respectively (FIG. 14b and FIG. 14c ). Enhancedexpression of F-α-DG was further demonstrated by western blot with theantibody AF6868 (FIG. 14b ). Finally, functionality of theribitol-induced glycosylated α-DG was supported by laminin overlay assay(FIG. 14b ).

To evaluate whether administration of ribitol affects expression ofglycosyltransferases responsible for the synthesis of Core M3 glycan onalpha-dystroglycan, we measured levels of mutant FKRP and LARGEtranscripts by quantitative real-time PCR in cardiac muscle, limb muscleand diaphragm (FIG. 22b ). No statistically significant difference inFKRP and LARGE transcript levels was observed between treated anduntreated samples in any of the tissues, suggesting that the effect ofribitol on levels of F-α-DG is independent to expression levels of theglycosyltransferases.

5% Ribitol treatment alleviates dystrophic pathology in P448L mice andimproves respiratory function. Therapeutic effect of 3 and 6 monthtreatments with 5% ribitol on dystrophic pathology of skeletal muscleswas demonstrated by histology. Hematoxylin and eosin (H&E) stainingshowed the large areas of degenerating fibers, high variation in fibersizes and high percentage of centrally nucleated fibers (CNF) in theskeletal muscles of the untreated P448L mice (FIG. 15a , FIG. 23a andFIG. 24). This was associated with focal inflammatory infiltrates.Treatment with ribitol improved the dystrophic pathology of limb musclesas evidenced by the diminished foci of necrotic fibers and a morehomogenously distributed fiber size. Quantitative analysis from TA andquadriceps showed a statistically significant decrease in the number offibers with small diameters (newly regenerated) indicating a decrease indegeneration after both 3 and 6-month ribitol treatments (FIG. 15b andFIG. 23b for TA and quadriceps, respectively). Furthermore, both 3 and 6month ribitol treatments significantly decreased areas of fibrotictissue detected by Masson's Trichrome staining when compared tountreated mice (FIG. 16a and FIG. 16b ). No significant difference inpercentage of CNF was observed between ribitol-treated and untreatedP448L mice (FIG. 15c and FIG. 23c for TA and quadriceps, respectively).This is expected as significant CNF reduction could only be achievedwith high dosage of viral particles with AAV gene therapy in the samemouse model.

Importantly, 5% ribitol treatment significantly reduced pathology of thediaphragm. Large foci of degenerating fibers were common in theuntreated diaphragms but became rarely observed in all the mice after 3and 6 month ribitol treatments (FIG. 15a and FIG. 24). The most strikingimprovement was the degree of fibrosis. The diaphragm of the untreatedmice showed heavy fibrosis at the 3 month time point (28.6% of tissuecross-section area), reaching more than 40% 6 months after the studyinitiation (FIG. 16a , FIG. 16b , and FIG. 25). However, the amount offibrotic tissues in the ribitol-treated cohorts was significantlyreduced to 11% and 18% after 3 and 6 month treatment, respectively.

The cardiac muscle of the P448L mice has limited pathology with only asmall increase in fibrotic area as disease progresses. H&E staining didnot show infiltration and degenerating fibers in both theribitol-treated and the untreated mice (FIG. 15a ). However, asignificant reduction in fibrotic area was observed in the cardiacmuscle of both 3 and 6 month ribitol-treated groups when compared to theuntreated (FIG. 16a and FIG. 16b ).

The significant improvement in histology of diaphragm with ribitoltreatment was associated with improvement in respiratory function shownby whole-body plethysmography at 3-months and 6-months post-initiationof the treatment. A trend of improvement was observed in tidal volume(TV), expiratory volume (EV) and minute volume (MV) in both 3 and 6month 5% ribitol-treated groups compared to the untreated P448L mice(FIG. 26a ). Importantly, the improvement in EV and MV after 6 monthribitol treatment became statistically significant. Improvement was alsoobserved in peak inspiratory flow (PIF) and peak expiratory flow (PEF)in the 6 month ribitol-treated group. However, significant improvementin limb muscle function was not demonstrated (FIG. 26b ). Overall, theseresults showed that 5% ribitol oral treatment is able to enhanceexpression of F-α-DG with significant improvement in pathology of allmuscles and in respiratory function.

Early treatment with 10% ribitol significantly improves skeletal musclefunction. We reported recently that therapeutic outcome throughrestoration of F-α-DG depends on earlier treatment in the P448L mice.Specifically, significant improvement in skeletal muscle functions byAAV gene therapy is achieved before the onset of the disease, but not inadult mutant mice when disease phenotype is already well established. Toassess whether early intervention with a higher dose of ribitol canachieve significant improvement in skeletal muscle functions, weinitiated a treatment with 10% ribitol in drinking water to the breedingfemales when they became pregnant, and continued the treatment to thepups until they reached 19 weeks of age. All the functional tests wereperformed at the same age as the cohort treated with 5% ribitol from7-week old for 3 months and age-matched untreated controls. Expressionof F-α-DG was detected in all skeletal muscles and in the cardiac muscleof the 10% ribitol-treated mice by immunohistochemistry (FIG. 17a ).F-α-DG was highly homogeneous in the cardiac muscle. Importantly, F-α-DGwas clearly detected with even distribution in the skeletal musclesincluding the diaphragm. Expression of F-α-DG was clearly detected bywestern blots with the IIH6C4, reaching 14%, 18% and 26% normal levelsin the heart, diaphragm and limb muscle respectively (FIG. 17b and FIG.17c ). Enhanced expression of F-α-DG was also demonstrated by westernblot with the antibody AF6868 (FIG. 17b ). Finally, functionality of theribitol-induced glycosylated α-DG was supported by laminin overlay assay(FIG. 17b ).

Consistent with the enhancement on the biochemical marker, dystrophicpathology in the 10% ribitol-treated mice was greatly alleviated withsignificantly fewer CNFs (FIG. 18a ). Most fibers of the limb muscleswere highly homogenous in shape and size and only a proportion of fiberswere centrally nucleated within the diseased muscles. Notably,improvement in pathology with reduced infiltration and fiber sizevariation was also observed in the diaphragm (FIG. 27). Furthermore,reduction in fibrosis was significant in cardiac muscle, and mostprominent in the diaphragm (FIG. 18b ).

Importantly, early treatment with 10% ribitol significantly improvedskeletal muscle functions of the P448L mice. Treadmill tests showed thatboth running distance and time of the treated mice were significantlylonger than the age-matched untreated mice (FIG. 18c ). Grip strengthtests also showed significant improvement on forelimb force from theribitol-treated mice compared to the untreated (FIG. 18d ). Significantimprovement in respiratory functions was also demonstrated byplethysmography (FIG. 18e and FIG. 28a ). Similar to the mutant micetreated for 6 months with 5% ribitol, a trend of improvement in tidalvolume (TV), minute volume (MV), end-expiratory and end-inspiratorypause (EEP and EIP, respectively) was observed, with MV and EEP reachingsignificant difference between the 10% ribitol treated and the untreatedP448L mice (FIG. 18e ). Furthermore, improvement on expired volume (EV),relaxation time (RT) and enhanced pause (Penh) was also observed (FIG.28a ).

Effects of ribitol treatment on body weight and histology of liver,kidney and spleen. No significant difference in body weight was observedbetween the mice treated with 10% ribitol from the embryonic stage,those treated with 5% ribitol from 7 weeks of age, or the age-matcheduntreated P448L mice at all time points although treated female micewere slightly heavier than the controls (FIG. 28b ).

The effect of ribitol treatment the pharmacological concentration of 5%on histology of liver, kidney and spleen was also evaluated with H&Estaining. All tissues showed normal structure without degeneration andinflammation, and no difference was observed between the untreated andthe ribitol treated cohorts as illustrated in the FIG. 29a . We alsoperformed biochemical analyses of serum markers for liver functionincluding alkaline phosphatase (ALP), alanine transaminase (ALT), totalbilirubin (t-Bil), conjugated bilirubin (c-Bil), and unconjugatedbilirubin (unc-Bil). In addition, kidney function was evaluated byanalyses of urea (BUN) and creatinine (Crea) levels. No statisticalsignificance was observed between untreated and treated P448L mice.Levels of triglycerides (TRG) and glucose (GLU) were also similarbetween the two cohorts (FIG. 29b ).

Despite significant advances in understanding the causes and clinicalmanifestation of dystroglycanopathies, almost no progress has been madefor the treatment of the diseases including those caused by FKRPmutations. Currently, physical therapy and other clinic managementroutinely provided to patients only serve as palliative care. The onlyoption of pharmacological intervention available is glucocorticoidsteroids which are being used anecdotally based on reported benefitsfrom other muscular dystrophies, especially Duchenne muscular dystrophy(DMD). Experimental therapy with the aim to restore F-α-DG byAAV-mediated gene therapy has been reported with high efficacy inpreventing disease progression in mouse models. However, clinical trialsof the therapy for the diseases with such a wide range of phenotypes arechallenging and remain to be conducted. Therefore, there is an urgentneed for developing experimental therapies. Here we show that ribitol, anatural pentose alcohol present in some plants and animals andconsidered as a metabolic intermediate or end-product, can effectivelyrestore therapeutic levels of F-α-DG and, more importantly, amelioratedystroglycanopathy caused by the FKRP P448L mutation which is associatedwith severe CMD phenotype in clinic. Our results offer a potentiallysafe and effective new class of treatment for restoration of F-α-DG toFKRP dystroglycanopathies. This treatment could be applied incombination with other therapies, such as AAV gene therapy for higherefficacy by enhancing the function of FKRP transgene. The results alsoraise the potential of developing similar approaches for enhancingF-α-DG in cells of other diseases associated with aberrantO-mannosylation of α-DG. An example of such application is for cancersexhibiting reduced or lack of F-α-DG in association with invasion andmetastasis which can be inhibited by gene transfer-mediated upregulationof F-α-DG.

FKRP dystroglycanopathy affects respiratory and cardiac muscles even indiseases with mild defects in skeletal muscles. Failures in respiratoryand cardiac functions are the prime causes for the lethality of thediseases. Therefore, restoration of F-α-DG and improvement in cardiacand respiratory functions are critically important for life quality andlongevity of patients. Ribitol treatment enhances F-α-DG in both cardiacand diaphragm muscles which is often most severely affected. This leadsto significant improvements in the pathology of the diaphragm withstriking reduction in fibrosis which may explain the enhancement ofrespiratory functions. Cardiac defects in both pathology and functionsin the P448L mice are limited and significant improvement in function isdifficult to demonstrate even with effective AAV9 gene therapy.Nevertheless, ribitol treatment is able to produce sustained andhomogenous expression of F-α-DG in the treated cardiac muscle, resultingin significant reduction in fibrosis. All the data therefore clearlydemonstrate therapeutic potential of the treatment to the two criticalorgans and their functions. Also important, ribitol treatment ofdifferent time frames up to 6 months shows no clear side effect. Oralribitol administration from pregnancy to adult of the P448L mice doesnot affect pregnancy, embryo development, body weight and overallbehavior of the mutant mice. These together with normal histology andlevels of serum markers for liver and kidney suggest the potential insafety for clinic applications.

Therapeutic effects of ribitol treatment are related to the enhancedF-α-DG. Ribitol supplementation has been shown to increase the levels ofCDP-ribitol both in cultured cells and in muscles of wild-type mice invivo. Our detection of increased levels of CDP-ribitol in both cardiacand skeletal muscles of FKRP mutant mice is consistent with the earlierreport. We also demonstrated the increase in the levels of ribitol-5Pand CDP-ribitol which is considered the substrate of ISPD, indicatingthat ribitol can be effectively converted to CDP-ribitol in both FKRPmutant muscle tissues as well as normal tissues. FKRP function isconsidered essential for functional glycosylation of α-DG. It isintelligible that the enhanced expression of F-α-DG in the diseasedmuscles also requires function of FKRP as in normal muscles. Indeed, ithas long been demonstrated that diseased muscles with missense mutationsof the FKRP gene remain capable of producing F-α-DG but at variablelower levels (hypoglycosylation). This is most convincingly demonstratedin mouse models with FKRP mutations. The mutant mice with common L276Imutations express low but clearly detectable levels of F-α-DG in bothskeletal and cardiac muscles. Considerable amount of F-α-DG is alsodetected in all compound heterozygotes containing L276I allele.

Despite the lack of expression of F-α-DG in the majority of musclefibers of the P448L mice, individual muscle fibers are able to expressnear normal levels of F-α-DG with laminin-binding capacity. Normallevels of F-α-DG are expressed in all regenerating fibers. More directevidence came from AAV-mediated expression of the P448L mutant FKRP,which is able to restore F-α-DG and protect muscles from degeneration.Interestingly, strong expression of F-α-DG is detected in all new bornskeletal and cardiac muscles of the P448L mice and this expression isnot associated with clear increase in mutant FKRP expression. Thisdemonstration supports the functionality of the mutant FKRPs and moreimportantly suggests that factor(s) other than FKRP expression couldcompensate for the reduced functionality of the mutant FKRPs.

We therefore hypothesize that the additional amount of ribitol allowsthe muscle fibers to produce higher than normal levels of FKRP substrate(CDP-ribitol) which enhances and partially compensates for the reducedfunction of mutant FKRPs (FIG. 19). Fortunately, more than 90% ofpatients with FKRP mutations contain at least one allele of the L276Imutation which retains at least partial function as demonstrated inpatient muscles and in the mutant mouse models. Therefore, this low costand easy to administer experimental therapy will likely be applicable tothe majority of patients with FRKP mutations. This, together with thenature of the drug, expected to be of limited side effects, would makeexecution of an early clinical trial much simpler

Animal care. All animal studies were approved by the InstitutionalAnimal Care and Use Committee (IACUC) of Carolinas Medical Center. Allmice were housed in the vivarium of Carolinas Medical Center followinganimal care guidelines of the institute. Animals were ear tagged priorto group assignment. Food and water were available ad libitum during allphase of the study. Body weight was measured from 6 weeks to 19 weeks ofage.

Mouse model and experimental procedure. FKRP P448L mutant mice weregenerated by the McColl-Lockwood Laboratory for Muscular DystrophyResearch. The mice contain a homozygous missense mutation (c.1343C>T,p.Pro448Leu) in the FKRP gene with the floxed neomycin resistant (Ned)cassette removed from the insertion site. C57BL/6 (wild-type/C57) micewere purchased from Jackson Laboratory.

Ribitol was purchased from Sigma (A5502 Adonitol, ≥98%, Sigma, St.Louis) and dissolved in drinking water to the final concentration of 5%or 10%. P448L mice aged at 4 weeks were treated with 5% ribitol drinkingwater for 1 month and P448L mice aged at 7 weeks were treated with 5%ribitol drinking water for 3 months and 6 months. All the mice wererandomly assigned to either treatment or control groups. And a minimumnumber of 4 mice were used for each group. No animal was excluded. P448Lfemale breeders were given 10% ribitol in drinking water duringpregnancy, and pups continued to be treated with 10% ribitol in drinkingwater until they were euthanized at 19 weeks of age. Untreatedage-matched P448L and wild-type C57BL/6 mice were used as controls. Theanimals were terminated at the end of each treatment time point andtissues including heart, diaphragm, TA, quadriceps, liver, spleen andkidney were collected for analyses.

Immunohistochemical and western blot analysis. Tissues were dissectedand snap-frozen in dry-ice-chilled-2-methylbutane. Forimmunohistochemical detection of functionally glycosylated α-DG, 6 μm ofthickness cross sections of untreated and C57 control, as well astissues from treated cohorts were included in each slide. Slides werefirst fixed in ice cold Ethanol:Acetic acid (1:1) for 1 min, blockedwith 10% normal goat serum (NGS) in 1×Tris-buffer saline (TBS) for 30min at room temperature, and incubated overnight at 4° C. with primarymouse monoclonal antibody IIH6C4 (EMD Millipore) (1:500) against α-DG.Negative controls received 10% normal goat serum in 1×TBS only. Sectionswere washed and incubated with secondary AlexaFLuor 488 goat anti-mouseIgM (Invitrogen) (1:500) at room temperature for 2 hr. Sections werewashed and finally mounted with fluorescence mounting medium (Dako)containing 1×DAPI (4′,6′-diamidino-2-phenylindole) for nuclear stainingImmunofluorescence was visualized using an Olympus BX51/BX52fluorescence microscope (Opelco) and images were captured using theOlympus DP70 digital camera system (Opelco). Slides were examined in ablinded manner by the investigator.

For western blot analysis, tissues were homogenized in extraction buffer(50 mM Tris-HCl pH 8.0, 150 mM NaCl, and 1% Triton X-100), supplementedwith 1× protease inhibitor cocktail (Sigma-Aldrich). Proteinconcentration was quantified by Bradford assay (Bio-Rad DC proteinassay). Eighty μg of protein was loaded on an 4-15% Bio-Rad Mini-PROTEANTGX gel (Bio-Rad) and immunoblotted. Amount of total protein loaded forC57 mice was half of the amount loaded for the P448L mice.Nitrocellulose membranes (Bio-Rad) were blocked with 5% milk in 1×PBSfor 2 hr at room temperature and then incubated with the followingprimary antibodies overnight at 4° C.: IIH6C4 (1:2000), AF6868 (R&DSystems) (1:1000) and α-actin (Sigma) (1:1000). Appropriate horseradishperoxidase (HRP)-conjugated secondary antibodies were incubated for 2 hrat room temperature. All blots were developed byelectrochemiluminescence immunodetection (PerkinElmer). For IIH6C4 bandquantification from western blot ImageJ software was used. For lamininoverlay assay, nitrocellulose membranes were blocked with lamininoverlay buffer (10 mM ethanolamine, 140 mM NaCl, 1 mM MgCl2, and 1 mMCaCl₂, pH 7.4) containing 5% nonfat dry milk for 1 hr at 4° C. followedby incubation with laminin from Engelbreth-Holm-Swarm murine sarcomabasement membrane (L2020) (Sigma) at a concentration of 2 μg/mlovernight at 4° C. in laminin overlay buffer. Membranes were thenincubated with rabbit anti-laminin antibody (Sigma) (1:1500) followed bygoat anti-rabbit HRP-conjugated IgG secondary antibody (Santa CruzBiotechnology) (1:3000). Blots were saturated with Western-LightningPlus ECL (Perkin-Elmer) before exposure to and developing of GeneMateauto-radiographic film (VWR).

Histopathological and morphometric analysis. Frozen tissues wereprocessed for hematoxylin and eosin (H&E) and Masson's Trichromestaining following standard procedures. Muscle cross-sectional fiberequivalent diameter were determined from tibialis anterior andquadriceps stained with H&E using MetaMorph v7.7 Software (MolecularDevices). Percentage of centrally nucleated myofibers were manuallyquantified from the same tissue sections stained with H&E. Fibrotic arearepresented by blue staining in the Masson's Trichrome stained sectionswas quantified from heart, diaphragm, tibialis anterior and quadricepsusing ImageJ software. For all the morphometric analyses, a total of 300to 400 fibers from two representative 20× magnification images per eachmuscle per animal was used.

Quantitative reverse transcriptase PCR assay. Tissues were collectedfrom heart, diaphragm and tibialis anterior. RNA was extracted usingTRIzol (Invitrogen) following the supplied protocol. Final RNA pelletwas re-suspended in 20 μl RNAse-nuclease free water.

Final RNA concentration was determined using Nanodrop 2000c. One μg ofRNA was subsequently converted to cDNA using the High-CapacityRNA-to-cDNA™ Kit (Applied Biosystems) following the supplied protocol.cDNA was then used for quantitative real-time PCR using the mouseFKRP-FAM (Mm00557870_m1) and LARGE-FAM (Mm00521885_m1) taqman assay withprimer limited GAPDH-VIC (Mm99999915_g1) as the internal control andTaqMan® Universal Master Mix II, with UNG (Life Technologies).Quantitative real time PCR was run on the BioRad CFX96 Touch™ Real-TimePCR Detection System (BioRad) following the standard real time PCRconditions suggested for taqman assays. Results of FKRP and LARGEtranscript were calculated and expressed as 2{circumflex over ( )}-ΔΔCtand compared across tissues and animals.

Metabolite extraction from muscle tissues and LC/MS-MS analysis. Ribitolwas purchased from Sigma (A5502). Ribitol-5P and CDP-ribitol weresynthesized by Z Biotech (Aurora, Colo.). Muscle tissues were collected,and blinded samples were subjected to the following procedure. Thirty to80 μg of frozen tissue samples were homogenized with 400 μl ofMeOH:Acetonitrile (ACN) (1:1) and then centrifugated for 5 min at 10,000rpm. The supernatants were removed, transferred to individual wells of96-well plate and analyzed by LC/MS-MS. An Applied Biosystems Sciex 4000(Applied Biosystems; Foster City, Calif.) equipped with a Shimadzu HPLC(Shimadzu Scientific Instruments, Inc.: Columbia, Md.) and Leapauto-sampler (LEAP Technologies; Carrboro, N.C.) were used to detectribitol, ribitol-5P and CDP-ribitol from tissue samples and syntheticcompounds. The metabolites were separated on a silica gel column(Hypersil Silica 250×4.6 mm, 5 micron particle size) using solvent A:water, 10 mM NH₄OAc, 0.1% formic acid and solvent B: MeOH:ACN (1:1). Thefollowing gradient was used: 0-12 min, 5% buffer B; 13-14 mm, 95% bufferB, 15-17 min, 5% buffer B. Under these conditions, ribitol, ribitol-5Pand CDP-ribitol eluted at 8.3 min, 7.5 min and 8.9 min, respectively.The metabolites were analyzed using electrospray ionization massspectrometry operated in positive ion mode, ESI+. Compoundsconcentration in tissue samples were determined based on standard curvesprepared by serial dilutions (200-0.01 μM) of each of the compound inMeOH:ACN (1:1).

Cell culture and LC/MS-MS analysis of isotopically labeled metabolites.C2C12 mouse myoblast (ATCC, CRL-1772) were seeded and grown in DMEMGlutaMax medium (Gibco by Life Technologies) supplemented with 10% fetalbovine serum and 100 μg/ml penicillin-streptomycin. Differentiation intomyotubes was induced by replacing the growth media with DMEMsupplemented with 1 μM Insulin (Sigma), 2% heat-inactivated horse serum(Gibco by Life Technologies), 2.5 μM Dexamethasone (Sigma) and 5 mM¹³C₅-ribitol (Omicron Biochemicals, Inc., South Bend, Ind. USA) whencells reached confluence. Cells were harvested 5 days later and analyzedby LC/MS-MS for detection of ribitol (153.2→98.8 m/z), ribitol-5P(233.1→98.8 m/z), CDP-ribitol (538.1→324.1 m/z), ¹³C-ribitol(158.3→103.8 m/z), ¹³C-ribitol-5P (238.1.→98.8 m/z) and CDP-¹³C-ribitol(543.1.→324.1 m/z) as described above.

Muscle function tests. For treadmill exhaustion test, 17 and 30 weeksold mice were placed on the belt of a five-lane-motorized treadmill(LE8700 treadmill, Panlab/Harvard Apparatus, Barcelona, Spain) suppliedwith shock grids mounted at the back of the treadmill, which delivered a0.2 mA current to provide motivation for exercise. Initially, the micewere subjected to an acclimation period (time, 5 min; speed, 8 cm/s, and0° incline). Immediately after acclimation period, the test commencedwith speed increases of 2 cm/s every minute until exhaustion. The testwas stopped and the time to exhaustion was determined when the mouseremained on the shock grid for 5 s without attempting to re-engage thetreadmill. For grip force test, forelimb and hindlimb in peak torque wasmeasured by a grip strength meter (Columbus Instruments). For forelimbforce, the animal was held so that only the forelimb paws grasp thespecially designed mouse flat mesh assembly, and was pulled back fromthe tail until the grip was broken. The force transducer recorded thepeak force reached when the animal's grip is broken. For hindlimb force,an angled mesh assembly was used. Mice were allowed to rest on theangled mesh assembly, facing away from the meter with its hindlimbs atleast one-half of the way down the length of the mesh. The mouse tailwas pulled directly toward the meter and parallel to the mesh assembly.During this procedure, the mice resist by grasping the mesh with allfour limbs. Pulling toward the meter was continued until the hindlimbsreleased from the mesh assembly. Five successful hindlimb and forelimbforce measurements within 2 minutes were recorded. The average value wasused for analysis. Force was presented as values of KGF (kilogram-force)units normalized to bodyweights (gr) as “Units/gr”. The tests wereperformed 1 week before euthanasia.

Whole body plethysmography. Respiratory functional analysis inconscious, freely moving 18 and 31 weeks old mice were measured using awhole-body plethysmography technique. The plethysmograph apparatus (emkaTechnologies, Falls Church, Va.) was connected to a ventilation pump forthe purpose of maintaining a constant air flow, a differential pressuretransducer, a usbAMP signal amplifier, and a computer running EMKA iox2software with the respiratory flow analyzer module, which was used todetect pressure changes due to breathing and recording the transducersignal. An initial amount of 20 mL of air was injected and withdrawn viaa 20 mL syringe into the chamber for the purpose of calibration. Micewere placed inside the “free moving” plethysmograph chamber and allowedto acclimate for 5 min in order to minimize any effects of stressrelated changes in ventilation. Resting ventilation was measured for aduration of 15 min after the acclimation period. Body temperatures ofall mice were assumed to be 37° C. and to remain constant during theventilation protocol.

Statistical analysis. All data are expressed as mean±SEM unless statedotherwise. Statistical analyses were performed with GraphPad Prismversion 7.01 for Windows (GraphPad Software). Individual means werecompared using multiple t tests. Differences were considered to bestatistically significant at p≤0.05 (*).

That which is claimed is:
 1. A method of treating limb girdle musculardystrophy 2I (LGMD-2I) in a subject in need thereof, comprisingadministering to the subject an effective amount of a pharmaceuticalcomposition comprising ribitol, thereby treating the LGMD-2I in thesubject.
 2. The method of claim 1, wherein the subject has aloss-of-function mutation in a fukutin-related protein (FKRP) gene. 3.The method of claim 1, wherein the pharmaceutical composition comprises5% ribitol.
 4. The method of claim 1, wherein the pharmaceuticalcomposition comprises 10% ribitol.
 5. The method of claim 1, wherein thepharmaceutical composition is administered orally.
 6. The method ofclaim 1, wherein the method reduces muscle weakness in the subject. 7.The method of claim 1, wherein the method increases ribitol-5P levels inmuscle tissue of the subject.
 8. The method of claim 1, wherein themethod increases CDP-ribitol levels in muscle tissue of the subject. 9.The method of claim 1, wherein the method increases α-dystroglycan(α-DG) glycosylation in muscle tissue of the subject.
 10. The method ofclaim 1, wherein the method comprises administering the pharmaceuticalcomposition for at least 30 days.
 11. The method of claim 1, wherein themethod comprises administering the pharmaceutical composition for atleast 1 month.
 12. The method of claim 1, wherein the method comprisesadministering the pharmaceutical composition for at least 3 months. 13.The method of claim 1, wherein the method comprises administering thepharmaceutical composition for at least 6 months.
 14. The method ofclaim 10, wherein the subject has a loss-of-function mutation in afukutin-related protein (FKRP) gene.
 15. The method of claim 10, whereinthe pharmaceutical composition comprises 5% ribitol.
 16. The method ofclaim 10, wherein the pharmaceutical composition comprises 10% ribitol.17. The method of claim 10, wherein the pharmaceutical composition isadministered orally.
 18. The method of claim 2, wherein the methodcomprises administering the pharmaceutical composition for at least 1month.
 19. The method of claim 2, wherein the method comprisesadministering the pharmaceutical composition for at least 3 months. 20.The method of claim 2, wherein the method comprises administering thepharmaceutical composition for at least 6 months.